Methods for improving a binding characteristic of a molecule

ABSTRACT

The present invention relates to methods for improving a binding characteristic of a molecule, e.g., a peptide, for a binding target, in which the molecule is covalently linked to a detectable moiety, e.g., an enzyme, or an active portion or derivative thereof. The present invention also relates to molecules produced by the methods of the present invention.

1. FIELD OF THE INVENTION

The present invention relates to methods for improving a bindingcharacteristic of a molecule, e.g., a peptide, for a binding target, inwhich the molecule is joined to a detectable moiety, e.g., an enzyme, orthe active portion thereof. The present invention also relates tomolecules produced by the methods of the present invention.

2. BACKGROUND OF THE INVENTION

Molecules that bind a particular target are useful in a number ofapplications, including diagnostic and therapeutic methods, affinitypurification methods, methods of delivery to specific locations, etc. Ofparticular interest are proteins and peptides, including antibodies,that bind particular and specific targets, for example, nucleic acids orproteins. Molecules that have particular binding abilities can begenerated in a number of ways. One method is by immunizing an animalwith a target molecule and subsequently isolating antibodies, orfragments thereof, that bind the target. Another method is by usingphage display or other display methods to isolate binding molecules,including proteins. See, e.g., Chen et al., 2001, Nat. Biotechnol.19:537-42.

In many cases, the binding properties of the isolated molecules obtainedby such methods are not ideal for their ultimate application. Severalmethods have been described to improve the binding properties, whichmostly involve generating variants of the starting sequence andidentifying variants with improved binding properties. See, e.g. Yang etal., 1995, J. Mol. Biol. 254:392-403; Schier et al., 1996, J. Mol. Biol.263:551-67; and Beiboer et al., 2000, J. Mol. Biol. 296:833-49, whichare related to phage display-based methods. However, such methods aretime consuming and require several rounds of “panning”. Further, it isknown that such methods can result in the enrichment of bindingmolecules that show reduced binding affinity for the selected target.Thus, typically, tens of thousands of potential molecules must bescreened to isolate those with improved binding ability, and thisscreening process typically requires the use of helper reagents, such asanti-phage antibodies and antibody-enzyme conjugates, that limit thesensitivity and precision of subsequent screens.

Another frequently used method for screening is the ELISA method. Inthis method, a binding target is attached to a surface (e.g., a well ina microtiter dish). The target attached to the surface is incubated witha candidate binding molecule in a first binding reaction. The firstbinding reaction is washed to remove unbound candidate molecules. Ahelper reagent (e.g., an antibody-enzyme conjugate) is then added for asecond binding reaction. The helper reagent binds the candidatemolecules bound to the target. After a second wash to remove unboundhelper reagent, a substrate is added. The substrate is converted into adetectable form by helper reagent bound to candidate binding moleculesbound to target.

The ELISA methodology has several drawbacks, principally due to therequirement of a second binding reaction. During the second bindingreaction, the helper reagent can interact non-specifically with thetarget or reaction vessel thus leading to a high background signal,which limits the ability to detect weakly bound molecules.

Other assay formats are also used to measure or detect bindinginteractions, including radioimmune and biotinylation-based bindingassays. Similarly, these assays also require helper reagents and sufferfrom the same limitations.

Thus, there remains in the art a need for more sensitive and efficientmethods for identifying molecules with improved binding characteristics.

Citation of a reference in this or any section of the specificationshall not be construed as an admission that such reference is prior artto the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for improvinga binding characteristic of a binding molecule, e.g., a peptide bindingsequence, in which the binding molecule is joined to a reportermolecule, e.g., an enzyme, or the active portion or catalytic domainthereof. The reporter molecule, and thus a binding molecule linked toit, can be detected without a second binding reaction, e.g., of the typeused in standard ELISA assays, as illustrated in FIG. 4.

In one aspect, the present invention provides methods of improving abinding characteristic, e.g., affinity, selectivity, release rate orturnover rate, of a prototype binding molecule for a target, comprising:contacting the target with a reporter fusion under conditions that allowthe reporter fusion to bind to the target, wherein the reporter fusioncomprises a reporter molecule and a variant binding molecule derivedfrom the prototype binding molecule that binds to the target, andselecting the reporter fusion if it binds the target with an improvedbinding characteristic relative to that of the prototype binding domainfor the target.

In another aspect, the invention provides methods for improving abinding characteristic of a binding molecule for a target, comprising:contacting the target with a library comprising a multiplicity ofreporter fusions, under conditions that allow a reporter fusion to bindthe target, wherein said reporter fusions comprise a reporter moleculeand a variant binding molecule derived from a prototype binding moleculethat binds the target, and selecting a reporter fusion that binds thetarget and has a binding characteristic for the target that is improvedrelative to the binding characteristic of the prototype binding moleculefor the target.

In another aspect, the invention provides methods for improving abinding characteristic of a binding molecule for a target, comprising:contacting a target with a library comprising a multiplicity of reporterfusions, under conditions that allow a reporter fusion to bind thetarget, wherein said reporter fusions comprise a reporter molecule and avariant binding molecule derived from a prototype binding molecule thatbinds the target, selecting a reporter fusion bound to the target andhaving a binding characteristic that is improved relative to the bindingcharacteristic of the prototype binding molecule, and removing thereporter molecule from the selected reporter fusion.

In one embodiment, the selecting step comprises incubating the reporterfusion bound to the target under conditions that cause a reporter fusionwith an undesirable binding characteristic to dissociate from thetarget. In another embodiment, the selecting step comprises incubatingthe reporter fusion bound to the target with multiple rounds ofconditions that cause a reporter fusion with an undesirable bindingcharacteristic to dissociate from the target, wherein each subsequentround of conditions causes dissociation of a reporter fusion with abetter binding characteristic for the target than the previous round. Inanother embodiment, the amount of bound reporter fusion is measuredbetween one or more rounds of dissociation. In another embodiment, theselecting step comprises selecting a reporter fusion if it binds to thetarget under a first condition better than it binds to the target undera second condition. In another embodiment, the condition is pH, with thefirst condition being a pH lower than the second condition. In anothersuch embodiment, the first condition is a pH higher than the secondcondition. In another embodiment, the condition is temperature, with thefirst condition being a lower temperature than the second condition. Inanother embodiment, the first condition is a temperature higher than thesecond condition. In another embodiment, the selecting step comprisesincubating the reporter fusion bound to the target in the presence ofproteases or under conditions that degrade or destabilize the reporterfusion. In another embodiment, conditions may include, but are notlimited to, heat, pH or subjugation to solutes that affect stability.

In another embodiment, the method further comprises repeating thecontacting and selecting steps, wherein the variant binding moleculeselected in a previous selection step is the prototype binding moleculeof a subsequent contacting step.

In another embodiment, the reporter molecule is a reporter sequence. Inanother embodiment, the reporter sequence is an enzyme or a functionalfragment or derivative of an enzyme. In another embodiment, the enzymeis a β-lactamase, β-galactosidase, phosphatase, peroxidase, reductase,esterase, hydrolase, isomerase or protease.

In another embodiment, the reporter fusion is selected if it has abinding affinity that is greater than the binding affinity of theprototype binding molecule. In another embodiment, the reporter fusionis selected if it has a binding affinity that is less than the bindingaffinity for the target of the prototype binding molecule. In anotherembodiment, the reporter fusion is selected if it has a bindingselectivity that is greater than the binding selectivity of theprototype binding molecule. In another embodiment, the reporter fusionis selected if it has a binding selectivity that is less than thebinding selectivity of the prototype binding molecule. In anotherembodiment, the reporter fusion is selected if it has a release ratethat is greater than the release rate of the prototype binding molecule.In another embodiment, the reporter fusion is selected if it has arelease rate that is less than the release rate of the prototype bindingmolecule. In another embodiment, the reporter fusion is selected if ithas a turnover rate that is greater than the turnover rate of theprototype binding molecule. In another embodiment, the reporter fusionis selected if it has a turnover rate that is less than the turnoverrate of the prototype binding molecule.

In another embodiment, the prototype binding molecule binds the targetwith a K_(d) of about 100 μM or less, 10 μM or less, 1 μM or less, 100nM or less, about 90 nM or less, about 80 nM or less, about 70 nM orless, about 60 nM or less, about 50 nM or less, about 40 nM or less,about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5nM or less, about 1 nM or less or about 0.1 nM or less. In yet anotherembodiment, the selected variant sequence binds the target with a K_(d)of about 100 μM or less, 10 μM or less, 1 μM or less, 100 nM or less,about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60nM or less, about 50 nM or less, about 40 nM or less, about 30 nM orless, about 20 nM or less, about 10 nM or less, about 5 nM or less,about 1 nM or less or about 0.1 nM or less. In another embodiment, theselected variant sequence binds the target with a K_(d) of about 100 μMor more, 10 μM or more, 1 M or more, 100 nM or more, about 90 nM ormore, about 80 nM or more, about 70 nM or more, about 60 nM or more,about 50 nM or more, about 40 nM or more, about 30 nM or more, about 20nM or more, about 10 nM or more, about 5 nM or more, about 1 nM or moreor about 0.1 nM or more.

In another embodiment, the variant binding molecule has been covalentlymodified relative to the prototype binding molecule.

In another embodiment, the binding molecule is a binding sequence. Inanother embodiment, the variant binding sequence has an amino acidsequence that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 or99% identical to the amino acid sequence of the prototype bindingsequence. In another embodiment, the variant binding sequence has beenpost-translationally modified relative to the prototype bindingsequence.

In another aspect, the present invention provides a binding moleculeproduced or identified by the methods of the present invention.

The present invention can be more fully explained by reference to thefollowing drawings, detailed description and illustrative examples.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of the present invention.

FIG. 2 illustrates a reporter fusion and a target of the invention.

FIG. 3 illustrates an embodiment of the invention by which variantbinding sequences are selected on the basis of having a particulardissociation constant that corresponds to tighter binding and a slowdissociation of the binding sequence and the target.

FIG. 4 illustrates certain differences between a method of the presentinvention and a standard ELISA assay.

FIG. 5 illustrates an embodiment of the invention for selectingpH-dependent binding sequences.

FIG. 6 illustrates an embodiment of the invention for screening reporterfusions on a surface.

FIG. 7 illustrates an amino acid sequence of β-lactamase.

FIG. 8 presents a schematic diagram of plasmid pADEPT06. P lac=lacpromoter, Pel B leader sequence=signal seq, L49VH=Heavy chain,L49VL=Light chain, 218 linker=linker region between heavy and lightchains, β-lactamase=β-lactamase gene, L49sFv-bl=scFv-BLA fusion,CAT=chloramphenicol resistance gene.

FIG. 9 shows results of a secondary screening of 21 mutants inquadruplicates. The x-axis shows variant designation and the y-axisshows the performance index. A ratio of bound activity at T₁ vs. T₀ wascalculated for each mutant, and the performance index was calculated bydividing the ratio of mutant over parent, as shown in Table 3.

FIG. 10 present details related to plasmid pME27.1 FIG. 10A presents aschematic diagram of plasmid pME27.1. P lac=lac promoter, Pel B leadersequence=signal seq, CAB1scFv=single chain antibody, BLA=β-lactamasegene, CAT=chloramphenicol resistance gene, T7 terminator=terminator.FIG. 10B presents shows the sequence of CAB1-scFv, the CDRs andmutations chosen for combinatorial mutagenesis. FIG. 10C presents andnucleotide sequence of pME27.1 FIG. 10D shows the amino acid sequence ofCAB1 which shows, for example, the sequence of the heavy chain, thesequence of the linker, the sequence of the light chain and the sequenceof BLA.

FIG. 11 shows binding assays and SDS page results. Specifically, FIG.11A shows the binding of variants from library NA05; FIG. 11B displaysand SDS PAGE of stable CAB1-BLA variants of the NA05 library; FIG. 11Cshows binding of various isolates from NA06 to CEA.

FIG. 12 shows a comparison of vH and vL sequences of CAB1-scFv with apublished frequency analysis of human antibodies. Specifically, FIG. 12Ashows the observed frequencies of the five most abundant amino acids inalignment of human sequence in the heavy chain; FIG. 12B shows theobserved frequencies of the five most abundant amino acids in alignmentof human sequence in the light chain.

FIG. 13 shows screening results of NA08 library. The x-axis showsbinding at pH 7.4, and the Y-axis shows binding at pH 6.5. Clones thatwere chosen are represented by a square.

FIG. 14 shows positions that were chosen for combinatorial mutagenesis.

FIG. 15 shows pH-dependent binding of NA08 variants to immobilizedcarcinoembryonic antigen.

FIG. 16 shows a chromatogram for 18 hour old extract of ME27.1

FIG. 17 shows an SDS-PAGE for 18 hour extract of ME27.1

FIG. 18 shows a chromatogram for 26 hour old extract of ME27.1

FIG. 19 shows an SDS-PAGE for 26 hour extract of ME27.1

FIG. 20 shows a chromatogram for 26 hour extract for ME 27.1 (4-5 days)

FIG. 21 shows an SDS-PAGE for 26 hour old extract. Conditions: 4-12%Tris-Bis/MES/Reducing conditions.

FIG. 22 shows CAB1 purification using anion exchange and PBA.

5. DETAILED DESCRIPTION OF THE INVENTION

A “binding molecule,” unless otherwise stated, includes both “prototypebinding molecules” and “variant binding molecules,” for example, abinding sequence.

A “prototype binding molecule” is a molecule that has a measurablebinding affinity for a target of interest.

A “variant binding molecule” is a molecule that is similar to, butdifferent from, a prototype binding molecule. The difference can be, forexample, any difference in structure, including, e.g., the addition,deletion or substitution of one or more atoms, amino acid residues orfunctional groups.

A “binding sequence,” unless otherwise stated, includes both “prototypebinding sequences” and “variant binding sequences.”

A “prototype binding sequence” is a peptide, polypeptide or proteinsequence that has a measurable binding affinity for a target ofinterest.

A “variant binding sequence” is a peptide, polypeptide or proteinsequence that is similar to, but different from, a prototype bindingsequence. The difference can be, for example, any difference insequence, including, e.g., addition, substitution, and/or deletion ofone or more amino acids. The difference also can be or include any formof covalent modification, e.g., a post-translational modification.

A “target” is anything, or any combination of things, to which apeptide, polypeptide or protein can bind.

A “reporter molecule” is a molecule that can be detected independent ofits binding to a detectable molecule, for example, a labeled antibody orother reporter molecule-binding molecule. A reporter sequence is a typeof reporter molecule.

A “detectable molecule” is a macromolecule that may bind to a moleculeand may be used to detected a reporter molecule; a detectable moleculeis not a small molecule substrate.

A “reporter sequence” is a reporter molecule that comprises a peptide,polypeptide or protein sequence that can be detected independent of itsbinding to a detectable molecule, for example, a labeled antibody orother reporter-sequence binding molecule. The reporter sequence can be,for example, an enzyme, a catalytically active fragment or derivative ofa protein, or a labeled peptide, polypeptide or protein, e.g., afluorescently labeled or a radioactively labeled peptide, polypeptide orprotein.

A “reporter fusion” is a molecule having a binding molecule and areporter molecule that are bound to each other, e.g., covalently boundto each other. The reporter fusion can optionally comprise otherelements, for example, one or more linker molecules joining one or moreparts of the reporter fusion, e.g., a reporter molecule and a bindingmolecule. Examples of reporter fusions include chimeric polypeptides andtargeted enzymes as described in U.S. patent application Ser. Nos.10/022,073 and 10/022,097, both filed Dec. 13, 2001, and incorporatedherein by reference in their entireties.

A “binding characteristic” is a measure of the interaction of twomolecules. Examples of binding characteristics include affinity,selectivity, release rate, turnover rate, stability of a moleculenecessary for binding and purification of a molecule which hasadditional or other binding characteristics. Turnover may refer to invirto or in vivo internalization and/or degradation by a cell or tissueof any or all of the molecules engaged in the binding interaction thatrenders the molecules unavailable for binding. For example, a targetand/or binding molecule may be internalized by a cell and degradedintracellularly. Binding molecules and targets may also be degraded bycell-surface proteases. Alternatively, following internalization, any orall of the binding molecules may be exocytosed to the cell surface andbe accessible and available for binding interactions.

“Selectivity” describes the ability of a binding molecule todiscriminate between different targets. A binding molecule is said tohave high selectivity if it binds with significantly higher affinity toits intended target than to most other surfaces or molecules.

Unless otherwise noted, the term “protein” is used interchangeably herewith the terms “peptide” and “polypeptide,” and refers to a moleculecomprising two or more amino acid residues joined by a peptide bond.

The terms “cell”, “cell line”, and “cell culture” can be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primarytransformed cell and cultures derived from that cell without regard tothe number of transfers. All progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same functionality as screened for in the originallytransformed cell are included in the definition of transformants. Thecells can be prokaryotic or eukaryotic.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for procaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, positive retroregulatory elements (see, e.g., U.S. Pat.No. 4,666,848, incorporated herein by reference), and possibly othersequences. Eucaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

The term “expression clone” refers to DNA sequences containing a desiredcoding sequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. The term “expression system” refers to a host transformed withan expression clone. To effect transformation, the expression clone maybe included on a vector; however, the relevant DNA may also beintegrated into the host chromosome.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a protein, polypeptideor precursor.

The term “operably linked” refers to the positioning of the codingsequence such that control sequences will function to drive expressionof the protein encoded by the coding sequence. Thus, a coding sequence“operably linked” to control sequences refers to a configuration whereinthe coding sequences can be expressed under the direction of a controlsequence.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides. Theexact size will depend on many factors, which in turn depends on theultimate function or use of the oligonucleotide. Oligonucleotides can beprepared by any suitable method, including, for example, cloning andrestriction of appropriate sequences and direct chemical synthesis by amethod such as the phosphotriester method of Narang et al., 1979, Meth.Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979,Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucageet al., 1981, Tetrahedron Lett. 22:1859-1862; and the solid supportmethod of U.S. Pat. No. 4,458,066, each incorporated herein byreference. A review of synthesis methods is provided in Goodchild, 1990,Bioconjugate Chemistry 1(3):165-187, incorporated herein by reference.

The term “primer” as used herein refers to an oligonucleotide which iscapable of acting as a point of initiation of synthesis when placedunder conditions in which primer extension is initiated. Synthesis of aprimer extension product that is complementary to a nucleic acid strandis initiated in the presence of the requisite four different nucleosidetriphosphates and a DNA polymerase in an appropriate buffer at asuitable temperature. A “buffer” includes cofactors (such as divalentmetal ions) and salt (to provide the appropriate ionic strength),adjusted to the desired pH.

A primer that hybridizes to the non-coding strand of a gene sequence(equivalently, is a subsequence of the coding strand) is referred toherein as an “upstream” or “forward” primer. A primer that hybridizes tothe coding strand of a gene sequence is referred to herein as an“downstream” or “reverse” primer.

The terms “restriction endonucleases” and “restriction enzymes” refer toenzymes, typically bacterial in origin, which cut double-stranded DNA ator near a specific nucleotide sequence.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,asparagine, glutamine, serine, threonine, tyrosine), nonpolar sidechains (e.g. alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan, cysteine, glycine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Standardthree-letter or one-letter amino acid abbreviations are used herein.

The peptides, polypeptides and proteins of the invention can compriseone or more non-classical amino acids. Non-classical amino acids includebut are not limited to the D-isomers of the common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid (4-Abu), 2-aminobutyric acid (2-Abu), 6-amino hexanoic acid (Ahx), 2-amino isobutyric acid (2-Aib),3-amino propionoic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs ingeneral.

As used herein, a “point mutation” in an amino acid sequence refers toeither a single amino acid substitution, a single amino acid insertionor single amino acid deletion. A point mutation preferably is introducedinto an amino acid sequence by a suitable codon change in the encodingDNA. Individual amino acids in a sequence are represented herein as AN,wherein A is the standard one letter symbol for the amino acid in thesequence, and N is the position in the sequence. Mutations within anamino acid sequence are represented herein as A₁ NA₂, wherein A₁ is thestandard one letter symbol for the amino acid in the unmutated proteinsequence, A₂ is the standard one letter symbol for the amino acid in themutated protein sequence, and N is the position in the amino acidsequence. For example, a G46D mutation represents a change from glycineto aspartic acid at amino acid position 46. The amino acid positions arenumbered based on the full-length sequence of the protein from which theregion encompassing the mutation is derived. Representations ofnucleotides and point mutations in DNA sequences are analogous.

As used herein, a “chimeric” protein refers to a protein whose aminoacid sequence represents a fusion product of subsequences of the aminoacid sequences from at least two distinct proteins. A chimeric proteinpreferably is not produced by direct manipulation of amino acidsequences, but, rather, is expressed from a “chimeric” gene that encodesthe chimeric amino acid sequence.

The term “host immune response” refers to a response of a hostorganism's immune system to contact with an immunogenic substance.Specific aspects of a host immune response can include, e.g., increasedantibody production, T cell activation, monocyte activation orgranulocyte activation. Each of these aspects can be detected and/ormeasured using standard in vivo or in vitro methods.

The term “Ab” or “antibody” refers to polyclonal and monoclonalantibodies, an entire immunoglobulin or antibody or any functionalfragment of an immunoglobulin molecule that binds to the target antigen.Examples of such functional entities include complete antibodymolecules, antibody fragments, such as Fv, single chain. Fv,complementarity determining regions (CDRs), V_(L) (light chain variableregion), V_(H) (heavy chain variable region), and any combination ofthose or any other functional portion of an immunoglobulin peptidecapable of binding to target antigen.

The term “% sequence homology” is used interchangeably herein with theterms “% homology,” “% sequence identity” and “% identity” and refers tothe level of amino acid sequence identity between two or more peptidesequences, when aligned using a sequence alignment program. For example,as used herein, 80% homology means the same thing as 80% sequenceidentity determined by a defined algorithm, and accordingly a homologueof a given sequence has greater than 80% sequence identity over a lengthof the given sequence. Exemplary levels of sequence identity include,but are not limited to, 60, 70, 80, 85, 90, 95, 98 or 99% or moresequence identity to a given sequence.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,known to one of skill in the art and publicly available on the Internetat http:www.ncbi.nlm.nih.gov/BLAST/”. See also Altschul et al., 1990, J.Mol. Biol. 215: 403-10 (with special reference to the published defaultsetting, i.e., parameters w=4, t=17) and Altschul et al., 1997, NucleicAcids Res., 25:3389-3402. Sequence searches are typically carried outusing the BLASTP program when evaluating a given amino acid sequencerelative to amino acid sequences in the GenBank Protein Sequences andother public databases. The BLASTX program is preferred for searchingnucleic acid sequences that have been translated in all reading framesagainst amino acid sequences in the GenBank Protein Sequences and otherpublic databases. Both BLASTP and BLASTX are run using defaultparameters of an open gap penalty of 11.0, and an extended gap penaltyof 1.0, and utilize the BLOSUM-62 matrix. See Altschul, et al., 1997.

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

“Hit density” is the fraction of useful clones in a library.

“Hapaxomer” is a restriction endonuclease that generates unique ends.See Berger, S. L. Anal Biochem 222:1 (1994).

The present invention relates to methods and compositions foridentifying variants of a binding molecule that have improved bindingproperties. The methods use reporter fusions comprising variant bindingmolecules and reporter molecules. Variants with improved bindingproperties are identified using the reporter molecule. The process canbe repeated multiple times. Ultimately, the binding molecule can beproduced without its reporter, either alone or as part of a largermolecule, e.g., a binding sequence that is part of a larger polypeptide.One embodiment of the method is illustrated in FIG. 1.

Reporter Fusions

A reporter fusion comprises a binding molecule operably linked to areporter molecule. The binding molecule and the reporter molecule areoperably linked if the binding molecule can bind the target and thereporter molecule can be detected. In one embodiment, the reporterfusion comprises a plurality of binding molecules. In anotherembodiment, the reporter fusion comprises a plurality of reportermolecules. In another embodiment, the reporter fusion comprises aplurality of binding molecules and a plurality of reporter molecules.

The binding molecule and the reporter molecule can be joined togetherusing any means for doing so provided that the binding molecule is ableto bind the target and the reporter molecule is detectable. In oneembodiment, the binding molecule and the reporter molecule arecovalently attached, for example, covalently attached to each otherdirectly (e.g., through a peptide bond or a disulfide bond), orcovalently attached to each other via a linker. Examples of linkersinclude peptides and peptide analogs (e.g., peptide nucleic acids),nucleic acids and nucleic acid analogs, and chemical cross linkers suchas p-azidobenzoyl hydrazide,N-(4-(p-azidosalicylamido)butyl)-3-(2′-pyridylthio)-propionamide,1-(p-azidosalicylamido0-4-(iodoacetamido)Butane,4-(p-azidosalicylamido)butylamine, 4,4′-diazidodiphenyl-ethane,4,4′-diazidodiphenyl-ether, dithio bis phenyl azide,bis(b-(4-azidosalicylaminoethyl)disulfide,sulfosuccinimidyl-6(4′-azido-2′-nitrophenylamino)hexanoate,sulfosuccinimidyl-4(p-azidophenyl)butyrate,sulfosuccinimidyl-(4-azidosalicylamido)hexanoate,N-hydroxysuccinimidyl-4-azido benzoate,N-hydroxysulfosuccinimidyl-4-azido benzoate,sulfosuccinimidyl-2-(p-azidosalicylamido)-ethyl-1,3-dithiopropionate,p-azidophenyl glyoxal, monohydrate,N-(4-(p-azidosalicylamido)butyl)-3-(2′-pyridylthio)-propionamide, and1-(p-azidosalicylamido0-4-(iodoacetamido)butane. In a more particularlydefined embodiment, the binding molecule is a binding sequence, thereporter molecule is a reporter sequence, and the reporter fusion is afusion protein. The fusion protein can be synthesized chemically, bydirect manipulation and joining of peptides, or translated in vivo or invitro from an appropriate nucleic acid template, as described below.Examples of reporter fusions that are fusion proteins are provided in,for example, Yamabhai et al., 1997, Anal. Biochem. 247:143-51;Schlehuber et al., 2001, Biophys. Chem. 96:213-28; Griep et al., 1999,Prot. Express. Purif. 16:63-69; Morino et al., 2001, J. Immunol. Meth.257:175-84; Wright et al., 2001, 253:223-32, incorporated herein byreference in their entireties.

In one aspect, the present invention provides a library comprising amultiplicity of reporter fusions. Various reporter fusions in thelibrary comprise a reporter sequence and a different variant bindingmolecule. The variant sequences are similar to, but different from, aprototype binding molecule. In one embodiment, the variant bindingmolecules are generated from a reporter fusion comprising the reportermolecule and the prototype binding molecule. In another embodiment, thereporter fusion comprises a polypeptide comprising a reporter sequenceand a binding sequence. In a more particularly defined embodiment,variant binding sequences are generated by mutating a nucleic acidencoding the reporter fusion. The mutagenesis can target all or part ofthe prototype binding sequence, and can alter the prototype bindingsequence in any way, including, for example, adding, deleting orsubstituting one or more amino acids. If more than one change is made,they can be made contiguously or in different parts of the prototypebinding sequence.

In another embodiment, the reporter fusion comprises the bindingsequence and/or the reporter sequence as an integral component. Thisapproach is useful for making diagnostic reagents or targeted enzymesthat have therapeutic (e.g., TEPT) or other applications. See, e.g.,U.S. patent application Ser. Nos. 10/022,073 and 10/022,097, both filedDec. 13, 2001, incorporated herein by reference in their entireties.

In another embodiment, the reporter fusion is made by grafting one ormore binding sequences into a reporter sequence, or by grafting one ormore reporter sequences into a binding sequence, e.g., as described incopending U.S. patent application Ser. Nos. 10/022,073 and 10/022,097,both filed Dec. 13, 2001, or in copending U.S. Pat. App. Ser. No.60,279,609 and U.S. Ser. No. 10,170,387 (attorney docket no. 9342-041and 40-999), incorporated herein by reference in their entireties.

Binding Molecules

A prototype binding molecule comprises a molecule that has a measurablebinding affinity for a target of interest. The prototype bindingmolecule can be any type of molecule, for example, a small organicmolecule, a biological molecule (e.g. a peptide, a polypeptide, aprotein, a nucleic acid, an oligonucleotide, a polynucleotide, a sugar,a metabolite, a lipid, a vitamin, a co-factor, a nucleotide or an aminoacid), a polymer, a drug, or an inorganic molecule. In one embodiment,the prototype binding molecule is a prototype binding sequence. Aprototype binding sequence comprises a peptide, either alone orcovalently attached to one or more other molecules, that binds to atarget. The peptide can have any amino acid sequence and can have one ormore covalent modifications. In one embodiment, the prototype bindingsequence is an antibody, antibody fragment, or derivative. In anotherembodiment, the prototype binding sequence is not an antibody, antibodyfragment, or derivative.

A variant binding molecule is similar to a prototype binding moleculethat binds a target but differs from it in one or more aspects. Thedifference can be any difference that affects a binding property of thebinding molecule. The difference can be, for example, one or moreinsertions, deletions and/or substitutions, or combinations thereof, ofatoms, amino acids or functional groups. In one embodiment, the bindingmolecule is a binding sequence, and the difference is a difference inthe amino acid sequence of the prototype binding sequence. The variantbinding sequence can be, for example, at least 50, 55, 60, 65, 70, 75,80, 85, 90, 95 , 98 or 99% identical to the prototype binding sequence.The amino acid sequence of the variant binding sequence can differ fromthe amino acid sequence of the prototype binding sequence by thepresence or absence of one or more non-classical amino acids or chemicalamino acid analogs. Non-classical amino acids include but are notlimited to the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid (4-Abu), 2-aminobutyric acid (2- Abu), 6-aminohexanoic acid (Ahx), 2-amino isobutyric acid (2-Aib), 3-amino propionoicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, and amino acid analogs in general.

In another embodiment, the variant binding sequences has or lacks acovalent modification relative to the prototype binding sequence, forexample, glycosylation, methylation, acetylation, phosphorylation,amidation, derivatization by protecting/blocking groups, proteolyticcleavage, etc., as well as any of other numerous chemical modifications,including but not limited to specific chemical cleavage by cyanogenbromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

These variant sequences can be rationally designed or can be generatedby random or semi-random insertions, deletions or substitutions.

The binding molecules of the invention can bind a target with anyaffinity, e.g., with a K_(d) of about 100 μM or less, 10 μM or less, 1μM or less, 100 nM or less, about 90 nM or less, about 80 nM or less,about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40nM or less, about 30 nM or less, about 20 nM or less, about 10 nM orless, about 5 nM or less, about 1 nM or less or about 0.1 nM or less. Inone embodiment, the affinity, or another binding characteristic, of abinding molecule for a target is dependent on the conditions under whichthe binding is conducted. Examples of conditions affecting bindinginclude pH, temperature, light, oxygen tension, salt concentration, thepresence or absence of binding co-factors, and other conditions found incopending U.S. Pat. App. Ser. No. 60/279,609 (attorney docket no.9342-042-999), filed concurrently with the present application,incorporated herein by reference in its entirety.

In one embodiment, the binding molecule is or is part of a targetedenzyme, e.g., as described in copending U.S. patent application Ser.Nos. 10/022,073 and 10/022,097, both filed Dec. 13, 2001, incorporatedherein by reference in their entireties.

In another embodiment, the binding molecule is or is part of amilieu-dependent binding molecule, e.g., a milieu-dependent targetedagent as described in copending U.S. Pat. App. Ser. No. 60/388,387(attorney docket no. 9342-042-999), filed concurrently with the presentapplication, incorporated herein by reference in its entirety.

In another embodiment, the binding molecule is or is part of amultifunctional polypeptide, e.g., as described in copending U.S. patentapplication Ser. No. 10/170,387 (attorney docket no. 9342-043-999),filed concurrently with the present application, incorporated herein byreference in its entirety.

Reporter Molecules

A reporter molecule can be any molecule that can be detected without thenecessity of being bound by a detectable molecule, e.g., a labeledantibody or other type of peptide or molecule that is labeled and bindsto the reporter molecule. The reporter molecule additionally can haveone or more desirable traits, for example, sensitive detection,selection of clones that produce a reporter fusion comprising thereporter molecule and a binding molecule of interest, stabilization ofthe reporter fusion or the binding molecule, protease-resistance of thereporter fusion or the binding sequence, easy purification, goodexpression or secretion of product into culture medium. Examples ofreporter molecules include radiolabeled substances, fluorescentmolecules, light-emitting molecules and molecules catalyzing orotherwise participating in a detectable chemical reaction, e.g., acolorimetric reaction.

In one embodiment, the reporter molecule is a reporter sequence. Inanother embodiment, the reporter sequence is an enzyme. The enzyme canbe any enzyme, or fragment or derivative of an enzyme, that can catalyzethe transformation of a substrate into a detectable reaction product.Examples of enzymes that can be used as reporter sequences includeβ-lactamases, β-galactosidases, phosphatases, peroxidases, reductases,esterases, hydrolases, isomerases and proteases.

In one embodiment, the reporter sequence is the enzyme β-lactamase(BLA). The enzyme is highly active towards the specific substratenitrocefin which allows the detection of bound reporter fusions at verylow concentrations. One can synthesize substrates with highersensitivity by using fluorogenic leaving groups. These substrates can bedesigned in analogy to BLA-activated prodrugs. See, e.g., Hudyma et al.,1993, Bioorg Med Chem Lett 3:323-28. BLA can be expressed in highconcentration in E.coli. In one embodiment, the present inventionprovides expression vectors that release substantial amounts of BLA intothe culture medium which greatly simplifies screening. BLA confersantibiotic resistance to its host. This can be exploited to quicklyevaluate the success of a cloning experiment. One can attach a bindingsequence to the N-terminus or C-terminus of BLA. If the mutagenesis ofthe binding sequence leads to sequences that are not correctlytranslated or that interfere with the cell physiology then suchundesirable mutants can in be rapidly identified or eliminated byselection with an appropriate antibiotic like cefotaxime orcarbenicillin.

BLA and it s fusion products can be easily purified by affinitychromatography using immobilized phenylboronic acid or similarinhibitors. See, e.g., Cartwright et al., 1984, Biochem J 221:505-12.

Using prodrugs that have been developed for cancer treatment it ispossible to select for cells that do not express BLA activity. This canbe used to identify mutants where the BLA gene has been inactivated.

In another embodiment, the BLA has a specific activity greater thanabout 0.01 U/pmol against nitrocefin using the assay described in U.S.patent application Ser. No. 10/022,097, filed Dec. 13, 2001,incorporated herein by reference in its entirety. In another embodiment,the specific activity is greater than about 0.1 U/pmol. In anotherembodiment, the specific activity is greater than about 1 U/pmol.

BLA enzymes are widely distributed in both gram-negative andgram-positive bacteria. BLA sequences are well known. A representativeexample of a BLA sequence is depicted in FIG. 7. BLA enzymes vary inspecificity, but have in common that they hydrolyze β-lactams, producingsubstituted β-amino acids. Thus, they confer resistance to antibioticscontaining β-lactams. Because BLA enzymes are not endogenous to mammals,they are subject to minimal interference from inhibitors, enzymesubstrates, or endogenous enzyme systems and therefore are particularlywell-suited for therapeutic administration. BLA enzymes are furtherwell-suited to the therapeutic methods of the present invention becauseof their small size (BLA from E. cloacae is a monomer of 43 kD; BLA fromE. coli is a monomer of 30 kD) and because they have a high specificactivity against their substrates and have optimal activity at 37° C.See Melton et al., Enzyme-Prodrug Strategies for Cancer Therapy, KluwerAcademic/Plenum Publishers, New York (1999).

The β-lactamases have been divided into four classes based on theirsequences. See Thomson et al., 2000, Microbes and Infection 2:1225-35.The serine β-lactamases are subdivided into three classes: A(penicillinases), C (cephalosporinases) and D (oxacillnases). Class Bβ-lactamases are the zinc-containing or metallo β-lactamases. Any classof BLA can be utilized to generate reporter sequence of the invention.

In one embodiment, the present invention provides a BLA reportersequence that comprises the sequence YXN at its substrate recognitionsite (throughout, “X” refers to any amino acid residue). In anotherembodiment, the BLA reporter sequence comprises the sequence RLYANASI atits active site. In another embodiment, the BLA reporter sequencecomprises a sequence at its active site that differs from the sequenceRLYANASI by one, two or three amino acid residues. The differences canbe, for example, the substitution of conservative amino acid residues,insertions, deletions and non-conservative amino acid substitutions.

In another embodiment, the present invention provides a BLA reportersequence that comprises the sequence KTXS at its substrate recognitionsite. In another embodiment, the BLA reporter sequence comprises thesequence VHKTGSTG at its active site. In another embodiment, the BLAreporter sequence comprises a sequence at its active site that differsfrom the sequence VHKTGSTG by one, two or three amino acid residues. Thedifferences can be, for example, the substitution of conservative aminoacid residues, insertions, deletions and non-conservative amino acidsubstitutions.

In another embodiment, the present invention provides a BLA reportersequence that comprises the sequences YXN and KTXS at its substraterecognition site. In another embodiment, the BLA reporter sequencecomprises the sequences VHKTGSTG and RLYANASI at its active site. Inanother embodiment, the BLA reporter sequence comprises sequences at itsactive site that differ from the sequences RLYANASI and VHKTGSTG by one,two or three amino acid residues. The differences can be, for example,the substitution of conservative amino acid residues, insertions,deletions and non-conservative amino acid substitutions.

In one embodiment, the BLA reporter sequnce comprises the amino acidsequence of FIG. 7. In another embodiment, the BLA reporter sequence isat least 50%, 60%, 70%, 80%, 90%, 95%, 98 or 99% or more identical tothe sequence depicted in FIG. 7.

In another embodiment, a nucleic acid encoding the BLA reporter sequencehybridizes to a nucleic acid complementary to a nucleic acid encodingthe amino acid sequence of FIG. 7 under highly stringent conditions. Thehighly stringent conditions can be, for example, hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel etal., eds., 1989, Current Protocols in Molecular Biology, Vol. I, GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., New York, atp. 2.10.3). Other highly stringent conditions can be found in, forexample, Current Protocols in Molecular Biology, at pages 2.10.1-16 andMolecular Cloning: A Laboratory Manual, 2d ed., Sambrook et al. (eds.),Cold Spring Harbor Laboratory Press, 1989, pages 9.47-57. In anotherembodiment, a nucleic acid encoding the BLA reporter sequence hybridizesto a nucleic acid complementary to a nucleic acid encoding the aminoacid sequence of FIG. 7 under moderately stringent conditions. Themoderately stringent conditions can be, for example, washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra). Othermoderately stringent conditions can be found in, for example, CurrentProtocols in Molecular Biology, Vol. I, Ausubel et al. (eds.), GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., 1989, pages2.10.1-16 and Molecular Cloning: A Laboratory Manual, 2d ed., Sambrooket al. (eds.), Cold Spring Harbor Laboratory Press, 1989, pages 9.47-57.

Fluorescent reporters like green fluorescent protein (GFP) or redfluorescent protein (RFP) also can be used.

In one embodiment, the reporter fusion comprises a plurality of reportermolecules, e.g., a plurality of reporter sequences. In a particularembodiment, a reporter fusion comprises a reporter sequence at itsN-terminus and at its C-terminus. A reporter fusion comprising aplurality of reporter sequences is particularly useful if the goal is toscreen for protease-resistant variants of a binding sequence. In oneembodiment, the reporter fusion comprises a BLA reporter sequence and afluorescent reporter sequence, e.g., GFP or RFP. The BLA reporter can beused for antibiotic selection and purification and the GFP reporter fordetection (e.g., using FACS).

Targets

The targets of the present invention can be any substance or compositionto which a molecule can be made to bind.

In one aspect, the target is a surface. In one embodiment, the surfaceis a biological surface. In another embodiment, the biological surfaceis a surface of an organ. In another embodiment, the biological surfaceis a surface of a tissue. In another embodiment, the biological surfaceis a surface of a cell. In another embodiment, the biological surface isa surface of a diseased organ, tissue or cell. In another embodiment,the biological surface is a surface of a normal or healthy organ, tissueor cell. In another embodiment, the surface is a macromolecule in theinterstitial space of a tissue. In another embodiment, the biologicalsurface is the surface of a virus or pathogen. In another embodiment,the surface is a non-biological surface. In another embodiment, thenon-biological surface is a surface of a medical device. In anotherembodiment, the medical device is a therapeutic device. In anotherembodiment, the therapeutic device is an implanted therapeutic device.In another embodiment, the medical device is a diagnostic device. Inanother embodiment, the diagnostic device is a well or tray.

Sources of cells or tissues include human, animal, bacterial, fungal,viral and plant. Tissues are complex targets and refer to a single celltype, a collection of cell types or an aggregate of cells generally of aparticular kind. Tissue may be intact or modified. General classes oftissue in humans include but are not limited to epithelial tissue,connective tissue, nerve tissue, and muscle tissue.

In another aspect, the target is a cancer-related target. Thecancer-related target can be any target that a composition of theinvention binds to as part of the diagnosis, detection or treatment of acancer or cancer-associated condition in a subject, for example, acancerous cell, tissue or organ, a molecule associated with a cancerouscell, tissue or organ, or a molecule, cell, tissue or organ that isassociated with a cancerous cell, tissue or organ (e.g., a tumor-bounddiagnostic or therapeutic molecule administered to a subject or to abiopsy taken from a subject, or a healthy tissue, such as vasculature,that is associated with cancerous tissue). Examples of cancer-relatedtargets are provided in U.S. Pat. No. 6,261,535, which is incorporatedherein by reference in its entirety.

The cancer-related target can be related to any cancer orcancer-associated condition. Examples of types of cancers includecarcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed typecancers.

In one embodiment, the cancer is a bone cancer, for example, Ewing'ssarcoma, osteosarcoma and rhabdomyosarcoma and other soft-tissuesarcomas. In another embodiment, the cancer is a brain tumor, forexample, oligodendroglioma, ependymoma, menengioma, lymphoma, schwannomaor medulloblastoma. In another embodiment, the cancer is breast cancer,for example, ductal carcinoma in situ of the breast. In anotherembodiment, the cancer is an endocrine system cancer, for example,adrenal, pancreatic, parathyroid, pituitary and thyroid cancers. Inanother embodiment, the cancer is a gastrointestinal cancer, forexample, anal, colorectal, esophogeal, gallbladder, gastric, liver,pancreatic, and small intestine cancers. In another embodiment, thecancer is a gynecological cancer, for example, cervical, endometrial,uterine, fallopian tube, gestational trophoblastic disease,choriocarcinoma, ovarian, vaginal, and vulvar cancers. In anotherembodiment, the cancer is a head and neck cancer, for example,laryngeal, oropharyngeal, parathryroid or thyroid cancer. In anotherembodiment, the cancer is a leukemic cancer, for example, acutelymphocytic leukemia, acute myelogenous leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, hairy cell leukemia, or amyeloproliferative disorder. In another embodiment, the cancer is a lungcancer, for example, a mesothelioma, non-small cell small cell lungcancer. In another embodiment, the cancer is a lymphoma, for example,AIDS-related lymphoma, cutaneous T cell lymphoma/mucosis fungoides,Hodgkin's disease, or non-Hodgkin's disease. In another embodiment, thecancer is metastatic cancer. In another embodiment, the cancer is amyeloma, for example, a multiple myeloma. In another embodiment, thecancer is a pediatric cancer, for example, a brain tumor, Ewing'ssarcoma, leukemia (e.g., acute lymphocytic leukemia or acute myelogenousleukemia), liver cancer, a lymphoma (e.g., Hodgkin's lymphoma ornon-Hodgkin's lymphoma), neuroblastoma, retinoblastoma, a sarcoma (e.g.,osteosarcoma or rhabdomyosarcoma), or Wilms' Tumor. In anotherembodiment, the cancer is penile cancer. In another embodiment, thecancer is prostate cancer. In another embodiment, the cancer is asarcoma, for example, Ewing's sarcoma, osteosarcoma, rhabdomyosarcomaand other soft-tissue sarcomas. In another embodiment, the cancer is askin cancer, for example, cutaneous T cell lymphoma, mycosis fungoides,Kaposi's sarcoma or melanoma. In another embodiment, the cancer istesticular cancer. In another embodiment, the cancer is thyroid cancer,for example, papillary, follicular, medullary, or anaplastic orundifferentiated thyroid carcinoma. In another embodiment, the cancer isurinary tract cancers, for example, bladder, kidney or urethral cancers.In another embodiment, the cancer or cancer-related condition isataxia-telangiectasia, carcinoma of unknown primary origin, Li-Fraumenisyndrome, or thymoma.

In another aspect, the cancer-related target is a molecule associatedwith a cancerous cell or tissue. In one embodiment, the molecule is atumor or tumor stroma antigen, for example, GD2, Lewis-Y, 72 kdglycoprotein (gp72, decay-accelerating factor, CD55, DAF, C3/C5convertases), CO17-1A (EpCAM, 17-1A, EGP-40), TAG-72, CSAg-P (CSAp), 45kd glycoprotein, HT-29 ag, NG2, A33 (43 kd gp), 38 kd gp, MUC-1, CEA,EGFR (HER1), HER2, HER3, HER4, HN-1 ligand, CA125, syndecan-1, Lewis X,PgP, FAP stromal Ag (fibroblast activation protein), EDG receptors(endoglin receptors), ED-B, laminin-5 (gamma2), cox-2 (+LN-5), PgP(P-glycoprotein), alphaVbeta3 integrin, alphaVbeta5, integrin, uPAR(urokinase plasminogen activator receptor), endoglin (CD105), folatereceptor osteopontin (EDG 1,3), p97 (melanotransferrin), farnesyltransferase or a molecule in an apoptotic pathway (e.g., a deathreceptor, fas, caspase or bcl-2) or a lectin.

In another aspect, the target is a hematopoietic cell. Hematopoieticcells encompass hematopoietic stem cells (HSCs), erythrocytes,neutrophils, monocytes, platelets, mast cells, eosinophils, basophils, Band T cells, macrophages, and natural killer cells. In one embodiment,the HSC has a surface antigen expression profile of CD34⁺ Thy-1⁺, andpreferably CD34⁺ Thy-1⁺ Lin⁻. Lin⁻ refers to a cell population selectedon the basis of the lack of expression of at least one lineage specificmarker. Methods for isolating and selecting HSCs are well known in theart and reference is made to U.S. Pat. Nos. 5,061,620, 5,677,136, and5,750,397, each of which is incorporated herein in its entirety.

In another aspect, the target is a molecule. In one embodiment, themolecule is an organic molecule. In another embodiment, the molecule isa biological molecule. In another embodiment, the biological molecule isa cell-associated molecule. In another embodiment, the cell-associatedmolecule is associated with the outer surface of a cell. In anotherembodiment, the cell-associated molecule is part of the extracellularmatrix. In another embodiment, the cell-associated molecule isassociated with the outer surface of a cell is a protein. In anotherembodiment, the protein is a receptor. In another embodiment, thecell-associated molecule is specific to a type of cell in a subject. Inanother embodiment, the type of cell is a diseased cell. In anotherembodiment, the diseased cell is a cancer cell. In another embodiment,the diseased cell is an infected cell. Other molecules that can serve astargets according to the invention include, but are not limited to,proteins, peptides, nucleic acids, carbohydrates, lipids,polysaccharides, glycoproteins, hormones, receptors, antigens,antibodies, toxic substances, metabolites, inhibitors, drugs, dyes,nutrients and growth factors.

In another aspect, the target is a surface feature, the surface featurecomprising two or more molecules. The two or more molecule may include,but are not limited to, proteins, peptides, nucleic acids,carbohydrates, lipids, polysacharrides, glycoproteins, hormones,receptors, antigens, antibodies, toxic substances, metabolites,inhibitors, drugs, dyes, nutrients or growth factors.

Non-limiting examples of protein and chemical targets encompassed by theinvention include chemokines and cytokines and their receptors.Cytokines as used herein refer to any one of the numerous factors thatexert a variety of effects on cells, for example inducing growth orproliferation. Non-limiting examples include interleukins (IL), IL-2,IL-3, IL-4 IL-6, IL-10, IL-12, IL-13, IL-14 and IL-16; soluble IL-2receptor; soluble IL-6 receptor; erythropoietin (EPO); thrombopoietin(TPO); granulocyte macrophage colony stimulating factor (GM-CSF); stemcell factor (SCF); leukemia inhibitory factor (LIF); interferons;oncostatin M (OM); the immunoglobulin superfamily; tumor necrosis factor(TNF) family, particularly TNF-α; TGFβ; and IL-1α; and vascularendothelial growth factor (VEGF) family, particularly VEGF (alsoreferred to in the art as VEGF-A), VEGF-B, VEGF-C, VEGF-D and placentalgrowth factor (PLGF). Cytokines are commercially available from severalvendors including Amgen (Thousand Oaks, Calif.), Immunex (Seattle,Wash.) and Genentech (South San Francisco, Calif.). Particularlypreferred are VEGF and TNF-α. Antibodies against TNF-α show thatblocking interaction of the TNF-α with its receptor is useful inmodulating over-expression of TNF-α in several disease states such asseptic shock, rheumatoid arthritis, or other inflammatory processes.VEGF is an angiogenic inducer, a mediator of vascular permeability, andan endothelial cell specific mitogen. VEGF has also been implicated intumors. Targeting members of the VEGF family and their receptors mayhave significant therapeutic applications, for example blocking VEGF mayhave therapeutic value in ovarian hyper stimulation syndrome (OHSS).Reference is made to N. Ferrara et al., (1999) Nat. Med. 5:1359 andGerber et al., (999) Nat. Med. 5:623. Other preferred targets includecell-surface receptors, such as T-cell receptors.

Chemokines are a family of small proteins that play an important role incell trafficking and inflammation. Members of the chemokine familyinclude, but are not limited to, IL-8, stomal-derived factor-1 (SDF-1),platelet factor 4, neutrophil activating protein-2 (NAP-2) and monocytechemo attractant protein-1 (MCP-1).

Other protein and chemical targets include, but are not limited to:immunoregulation modulating proteins, such as soluble human leukocyteantigen (HLA, class I and/or class II, and non-classical class I HLA (E,F and G)); surface proteins, such as soluble T or B cell surfaceproteins; human serum albumin; arachadonic acid metabolites, such asprostaglandins, leukotrienes, thromboxane and prostacyclin; IgE, auto oralloantibodies for autoimmunity or allo- or xenoimmunity, Ig Fcreceptors or Fc receptor binding factors; G-protein coupled receptors;cell-surface carbohydrates; angiogenesis factors; adhesion molecules;ions, such as calcium, potassium, magnesium, aluminum, and iron; fibrilproteins, such as prions and tubulin; enzymes, such as proteases,aminopeptidases, kinases, phosphatases, DNAses, RNAases, lipases,esterases, dehydrogenases, oxidases, hydrolases, sulphatases, cyclases,transferases, transaminases, carboxylases, decarboxylases, superoxidedismutase, and their natural substrates or analogs; hormones and theircorresponding receptors, such as follicle stimulating hormone (FSH),leutinizing hormone (LH), thyroxine (T4 and T3), apolipoproteins, lowdensity lipoprotein (LDL), very low density lipoprotein (VLDL),cortisol, aldosterone, estriol, estradiol, progesterone, testosterone,dehydroepiandrosterone (DHBA) and its sulfate (DHEA-S); peptidehormones, such as renin, insulin, calcitonin, parathyroid hormone (PTH),human growth hormone (hGH), vasopressin and antidiuretic hormone (AD),prolactin, adrenocorticotropic hormone (ACTH), LHRH,thyrotropin-releasing hormone (THRH), vasoactive intestinal peptide(VIP), bradykinin and corresponding prohormones; catechcolamines such asadrenaline and metabolites; cofactors including atrionatriutic factor(AdF), vitamins A, B, C, D, E and K, and serotonin; coagulation factors,such as prothrombin, thrombin, fibrin, fibrinogen, Factor VIII, FactorIX, Factor XI, and von Willebrand factor; plasminogen factors, such asplasmin, complement activation factors, LDL and ligands thereof, anduric acid; compounds regulating coagulation, such as hirudin, hirulog,hementin, hepurin, and tissue plasminigen activator (TPA); nucleic acidsfor gene therapy; compounds which are enzyme antagonists; and compoundsbinding ligands, such as inflammation factors; and receptors and otherproteins that bind to one or more of the preceding molecules.

Non-human derived targets include without limitation drugs, especiallydrugs subject to abuse, such as cannabis, heroin and other opiates,phencyclidine (PCP), barbiturates, cocaine and its derivatives, andbenzadiazepine; toxins, such as heavy metals like mercury and lead,arsenic, and radioactive compounds; chemotherapeutic agents, such asparacetamol, digoxin, and free radicals; bacterial toxins, such aslipopolysaccharides (LPS) and other gram negative toxins, Staphylococcustoxins, Toxin A, Tetanus toxins, Diphtheria toxin and Pertussis toxins;plant and marine toxins; snake and other venoms, virulence factors, suchas aerobactins, or pathogenic microbes; infectious viruses, such ashepatitis, cytomegalovirus (CMV), herpes simplex virus (HSV types 1, 2and 6), Epstein-Barr virus (EBV), varicella zoster virus (VZV), humanimmunodeficiency virus (HIV-1, -2) and other retroviruses, adenovirus,rotavirus, influenzae, rhinovirus, parvovirus, rubella, measles, polio,pararyxovirus, papovavirus, poxvirus and picornavirus, prions, plasmodiatissue factor, protozoans, such as Entamoeba histolitica, Filaria,Giardia, Kalaazar, and toxoplasma; bacteria, gram-negative bacteriaresponsible for sepsis and nosocomial infections such as E. coli,Acynetobacter, Pseudomonas, Proteus and Klebsiella, also gram-positivebacteria such as Staphylococcus, Streptococcus, Meningococcus andLlycobacteria, Chlamydiae Legionnella and Anaerobes; fungi such asCandida, Pneumocystis, Aspergillus, and Mycoplasma.

In one aspect the target includes an enzyme such as proteases,aminopeptidases, kinases, phosphatases, DNAses, RNAases, lipases,esterases, dehydrogenases, oxidases, hydrolases, sulphatases,cellulases, cyclases, transferases, transaminases, carboxylases,decarboxylases, superoxide dismutase, and their natural substrates oranalogs. Particularly preferred enzymes include hydrolases, particularlyalpha/beta hydrolases; serine proteases, such as subtilisins, andchymotrypsin serine proteases; cellulases; and lipases.

In another embodiment, the target is a non-biological material. Inanother embodiment, the non-biological material is a fabric. In anotherembodiment, the fabric is a natural fabric. In another embodiment, thefabric is cotton. In another embodiment, the fabric is silk. In anotherembodiment, the fabric is wool. In another embodiment, the fabric is anon-natural fabric. In another embodiment, the fabric is nylon. Inanother embodiment, the fabric is rayon. In another embodiment, thefabric is polyester. In another embodiment, the non-biological materialis a plastic. In another embodiment, the non-biological material is aceramic. In another embodiment, the non-biological material is a metal.In another embodiment, the non-biological material is rubber. In anotherembodiment, the non-biological material is wood.

In another embodiment the target is a microcircuit. This circuit can bein its finished form or in any stage of circuit manufacturing. See,e.g., van Zant, 2000, Microchip Fabrication, McGraw-Hill, New York,incorporated herein by reference in its entirety.

In another embodiment, the target is not an antibody (e.g., a polyclonalantibody, a monoclonal antibody, an scFv, or another antigen-bindingfragment of an antibody).

Methods of Selecting Variant Binding Molecules

In one aspect, the present invention provides methods of screeningreporter fusions comprising variant binding molecules and reportermolecules to identify binding molecules with desired bindingcharacteristics. Any method of screening reporter fusions comprisingvariant binding molecules and reporter molecules that can identifybinding molecules with improved binding characteristics can be used.

Reporter fusions can be used in various ways that allow one to assay fordifferent properties of the binding molecule. For instance, by allowingfor sufficient time between measurements to reach binding equilibriumone can identify variants with improved binding affinity. Alternatively,one can screen a population of variants under two or more differentconditions to identify variants that differentiate between varioustargets or variants that show differential binding in dependence of thereaction conditions. See, e.g., U.S. Pat. App. Ser. No. 60/388,387(attorney docket no. 9342-042-999), filed concurrently with the presentapplication, incorporated herein by reference in its entirety.

In one embodiment, the binding molecule is a binding sequence. Oneembodiment of a screen for selecting an improved binding sequence isillustrated in FIG. 3. The process starts with a population of clones,where various clones produce a different reporter fusion comprising adifferent variant of a prototype binding sequence. The clones arecultured under conditions that facilitate the expression of the reporterfusions. In one embodiment, reporter fusions are released by the clonesinto the culture medium. Subsequently, a part of the culture istransferred to a microtiter plate to which the target of interest hasbeen bound. After incubation to allow target-reporter fusioninteraction, unbound reporter fusion is removed, for example, by washingor filtration. Then a chromogenic substrate is added to determine thequantity of bound reporter fusion for each variant. During thismeasurement, a fraction of the bound reporter fusion can dissociate fromthe target. Subsequently, one can remove the dissociated reporter fusionand add fresh substrate to measure the remaining concentration of boundreporter fusion. This process of washing and measuring can be repeatedseveral times. As a result, one can determine the dissociation rate ofeach variant in the population and detect variants with improved bindingproperties.

FIG. 5 shows, as an example, a screen at two different pH values. Bycomparing the values obtained under both pH conditions one can identifyvariants that show pH-dependent binding to their target. Furtherexamples of methods of screening for binding sequences that bind to atarget better under a first set of conditions than they bind under asecond set of conditions are provided in copending U.S. Pat. App. Ser.No. 60/388,387 (attorney docket no. 9342-042-999), filed concurrentlywith the present application, incorporated herein by reference in itsentirety. In a similar way, one can compare binding in the presence ofdifferent effector molecules to obtain variants which showeffector-dependent binding to a target.

In another embodiment, an improved binding molecule is selected bycontacting a non-biological target with the reporter fusion, forexample, a computer chip at any stage during its manufacture, or afterit is manufactured, or a surface (e.g. glass, plastic, fabric, film ormembrane) exhibiting, e.g., coated with, a target molecule.

Several methods have been described for manufacturing arrays ofcompounds. These methods can be adapted to screen populations ofreporter fusions for binding to a target. One embodiment of this processis illustrated in FIG. 6. Aliquots of variants are transferred onto asurface (e.g., membrane, plastic or glass) which carries bound target.The target can be, for example, a molecule or a cell of interest.Unbound reporter fusions are removed by washing and a substrate is addedto detect the remaining bound reporter fusion. It is important to use asubstrate that can be used to detect surface-bound reporter fusion. Suchsubstrates are commonly used for immunohistochemistry, e.g.,5-bromo-4-chloro-2-indoyl β-D-galactopyranoside, diaminobenzidine, ELF®97 esterase substrate (Molecular Probes, Eugene, Oreg.), ELF® 97phosphatase substrate (Molecular Probes), ELF® 97 β-D-glucuronide, ELF®97 N-acetylglucosaminide.

In addition, one can use fluorescent reporters to screen for variantbinding sequences that bind to a target of interest, e.g., a cell. Inone embodiment, the assay comprises one or more of the following steps:

Generating population of reporter fusions in a suitable host;

Growing host clones to produce reporter fusions;

Mixing the reporter fusions with a cell suspension;

Adding a fluorescent reference protein that shows fluorescence that canbe distinguished from the fluorescent reporter; and

Analyzing each cell suspension in a fluorescence activated cell sorter(FACS) to identify clones of reporter fusions that differ in theirbinding behavior from the control protein.

In one embodiment, a variant binding sequence is selected byimmobilizing reporter fusion-producing cells in agarose beads or similarmaterial to which the target has been attached. The reporter fusion canbind to the target in the bead and it can be detected by, for example,using a fluoregenic substrate, which allows stained beads to be sortedusing a fluorescence-activated cell sorter (FACS). See, e.g., Gray etal., 1995, J. Immun. Meth. 182:155-63.

In another embodiment, the screening method comprises multiple rounds ofgenerating a variant binding molecule of a prototype binding molecule,contacting the target with a reporter fusion comprising the variantbinding molecule and a reporter molecule, and selecting the variantsequence if it binds to the target with a desired bindingcharacteristic, wherein the variant binding molecule selected in aprevious selection step is the prototype binding molecule of thesubsequent generating step. Using this approach, multiple rounds ofscreening can be used to select binding molecules with increasinglyrefined binding characteristics.

In another embodiment, the screening method comprises contacting thetarget with a library of reporter fusions, wherein various reporterfusions in the library comprise a different variant binding molecules.In a more particularly defined embodiment, the method comprises usingmultiple rounds of screening, as described herein, wherein in each roundthe target is contacted with a library of reporter fusions comprisingvariant binding molecules that are derived from the binding moleculeselected in a previous round.

Nucleic Acids and Methods of Making Reporter Sequences, BindingSequences and Reporter Fusions

In another aspect, the present invention provides a nucleic acidencoding a polypeptide comprising all or part of a reporter sequence, abinding sequence or a reporter fusion. The nucleic acid can be, forexample, a DNA or an RNA. The present invention also provides a plasmidcomprising a nucleic acid encoding a polypeptide comprising all or partof a reporter sequence, a binding sequence or a reporter fusion. Theplasmid can be, for example, an expression plasmid that allowsexpression of the polypeptide in a host cell or organism, or in vitro.The expression vector can allow expression of the polypeptide in, forexample, a bacterial cell. The bacterial cell can be, for example, an E.coli cell.

Because of the redundancy in the genetic code, typically a large numberof DNA sequences encode any given amino acid sequence and are, in thissense, equivalent. As described below, it may be desirable to select oneor another equivalent DNA sequences for use in a expression vector,based on the preferred codon usage of the host cell into which theexpression vector will be inserted. The present invention is intended toencompass all DNA sequences that encode the reporter sequence, bindingsequence or reporter fusion.

Production of the reporter sequence, binding sequence or reporter fusionof the invention can be carried out using a recombinant expressionclone. The construction of the recombinant expression clone, thetransformation of a host cell with the expression clone, and the cultureof the transformed host cell under conditions which promote expression,can be carried out in a variety of ways using techniques of molecularbiology well understood in the art. Methods for each of these steps aredescribed in general below. Preferred methods are described in detail inthe examples.

An operable expression clone is constructed by placing the codingsequence in operable linkage with a suitable control sequences in anexpression vector. The vector can be designed to replicate autonomouslyin the host cell or to integrate into the chromosomal DNA of the hostcell. The resulting clone is used to transform a suitable host, and thetransformed host is cultured under conditions suitable for expression ofthe coding sequence. The expressed reporter sequence, binding sequenceor reporter fusion is isolated from the medium or from the cells,although recovery and purification of the reporter sequence, bindingsequence or reporter fusion may not be necessary in some instances.

Construction of suitable clones containing the coding sequence and asuitable control sequence employs standard ligation and restrictiontechniques that are well understood in the art. In general, isolatedplasmids, DNA sequences, or synthesized oligonucleotides are cleaved,modified, and religated in the form desired. Suitable restriction sitescan, if not normally available, be added to the ends of the codingsequence so as to facilitate construction of an expression clone.

Site-specific DNA cleavage is performed by treating with a suitablerestriction enzyme (or enzymes) under conditions that are generallyunderstood in the art and specified by the manufacturers of commerciallyavailable restriction enzymes. See, e.g., product catalogs from Amersham(Arlington Heights, Ill.), Roche Molecular Biochemicals (Indianapolis,Ind.), and New England Biolabs (Beverly, Mass.). In general, about 1 μgof plasmid or other DNA is cleaved by one unit of enzyme in about 20 μlof buffer solution; in the examples below, an excess of restrictionenzyme is generally used to ensure complete digestion of the DNA.Incubation times of about one to two hours at a temperature which isoptimal for the particular enzyme are typical. After each incubation,protein is removed by extraction with phenol and chloroform; thisextraction can be followed by ether extraction and recovery of the DNAfrom aqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. See, e.g.,Maxam et al., 1980, Methods in Enzymology 65:499-560.

Restriction enzyme-cleaved DNA fragments with single-strand“overhanging” termini can be made blunt-ended (double-strand ends) by,for example, treating with the large fragment of E. coli DNA polymeraseI (Klenow) in the presence of the four deoxynucleoside triphosphates(dNTPs) using incubation times of about 15 to 25 minutes at 20° C. to25° C. in 50 mM Tris, pH 7.6, 50 mM NaCl, 10 mM MgCl₂, 10 mM DTT, and 5to 10 μM dNTPs. The Klenow fragment fills in at 5′ protruding ends, butchews back protruding 3′ single strands, even though the four dNTPs arepresent. If desired, selective repair can be performed by supplying oneor more selected dNTPs, within the limitations dictated by the nature ofthe protruding ends. After treatment with Klenow, the mixture isextracted with phenol/chloroform and ethanol precipitated. Similarresults can be achieved using S1 nuclease, because treatment underappropriate conditions with S1 nuclease results in hydrolysis of anysingle-stranded portion of a nucleic acid.

Ligations can be performed, for example, in 15-30 μl volumes under thefollowing standard conditions and temperatures: 20 mM Tris-Cl, pH 7.5,10 mM MgCl₂, 10 mM DTT, 33 μg/ml BSA, 10-50 mM NaCl, and either 40 μMATP and 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for ligation offragments with complementary single-stranded ends) or 1 mM ATP and0.3-0.6 units T4 DNA ligase at 14° C. (for “blunt end” ligation).Intermolecular ligations of fragments with complementary ends areusually performed at 33-100 μg/ml total DNA concentrations (5-100 nMtotal ends concentration). Intermolecular blunt end ligations (usuallyemploying a 20-30 fold molar excess of linkers, optionally) areperformed at 1 μM total ends concentration.

In vector construction, the vector fragment is commonly treated withbacterial or calf intestinal alkaline phosphatase (BAP or CIAP) toremove the 5′ phosphate and prevent religation and reconstruction of thevector. BAP and CIAP digestion conditions are well known in the art, andpublished protocols usually accompany the commercially available BAP andCIAP enzymes. To recover the nucleic acid fragments, the preparation isextracted with phenol-chloroform and ethanol precipitated to remove thephosphatase and purify the DNA. Alternatively, religation of unwantedvector fragments can be prevented by restriction enzyme digestion beforeor after ligation, if appropriate restriction sites are available.

Correct ligations for plasmid construction can be confirmed using anysuitable method known in the art. For example, correct ligations forplasmid construction can be confirmed by first transforming a suitablehost, such as E. coli strain DG101 (ATCC 47043) or E. coli strain DG116(ATCC 53606), with the ligation mixture. Successful transformants areselected by ampicillin, tetracycline or other antibiotic resistance orsensitivity or by using other markers, depending on the mode of plasmidconstruction, as is understood in the art. Plasmids from thetransformants are then prepared according to the method of Clewell etal., 1969, Proc. Natl. Acad. Sci. USA 62:1159, optionally followingchloramphenicol amplification. See Clewell, 1972, J. Bacteriol. 110:667.Alternatively, plasmid DNA can be prepared using the “Base-Acid”extraction method at page 11 of the Bethesda Research Laboratoriespublication Focus 5 (2), and very pure plasmid DNA can be obtained byreplacing steps 12 through 17 of the protocol with CsCl/ethidium bromideultracentrifugation of the DNA. As another alternative, a commerciallyavailable plasmid DNA isolation kit, e.g., HISPEED™, QIAFILTER™ andQIAGEN™ plasmid DNA isolation kits (Qiagen, Valencia Calif.) can beemployed following the protocols supplied by the vendor. The isolatedDNA can be analyzed by, for example, restriction enzyme digestion and/orsequenced by the dideoxy method of Sanger et al., 1977, Proc. Natl.Acad. Sci. USA 74:5463, as further described by Messing et al., 1981,Nuc. Acids Res. 9:309, or by the method of Maxam et al., 1980, Methodsin Enzymology 65:499.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, insect, or mammalian cells are used ashosts. Procaryotic hosts are in general the most efficient andconvenient for the production of recombinant proteins and are thereforepreferred for the expression of the protein.

The procaryote most frequently used to express recombinant proteins isE. coli. However, microbial strains other than E. coli can also be used,such as bacilli, for example Bacillus subtilis, various species ofPseudomonas and Salmonella, and other bacterial strains. In suchprocaryotic systems, plasmid vectors that contain replication sites andcontrol sequences derived from the host or a species compatible with thehost are typically used.

For expression of constructions under control of most bacterialpromoters, E. coli K12 strain MM294, obtained from the E. coli GeneticStock Center under GCSC #6135, can be used as the host. For expressionvectors with the P_(L)N_(RBS) or P_(L) T7_(RBS) control sequence, E.coli K12 strain MC1000 lambda lysogen, N₇N₅₃cI857 SusP₈₀, ATCC 39531,may be used. E. coli DG116, which was deposited with the ATCC (ATCC53606) on Apr. 7, 1987, and E. coli KB2, which was deposited with theATCC (ATCC 53075) on Mar. 29, 1985, are also useful host cells. For M13phage recombinants, E. coli strains susceptible to phage infection, suchas E. coli K12 strain DG98 (ATCC 39768), are employed. The DG98 strainwas deposited with the ATCC on Jul. 13, 1984.

For example, E. coli is typically transformed using derivatives ofpBR322, described by Bolivar et al., 1977, Gene 2:95. Plasmid pBR322contains genes for ampicillin and tetracycline resistance. These drugresistance markers can be either retained or destroyed in constructingthe desired vector and so help to detect the presence of a desiredrecombinant. Commonly used procaryotic control sequences, i.e., apromoter for transcription initiation, optionally with an operator,along with a ribosome binding site sequence, include the β-lactamase(penicillinase) and lactose (lac) promoter systems, see Chang et al.,1977, Nature 198:1056, the tryptophan (trp) promoter system, see Goeddelet al., 1980, Nuc. Acids Res. 8:4057, and the lambda-derived P_(L)promoter, see Shimatake et al., 1981, Nature 292:128, and gene Nribosome binding site (N_(RBS)). A portable control system cassette isset forth in U.S. Pat. No. 4,711,845, issued Dec. 8, 1987. This cassettecomprises a P_(L) promoter operably linked to the N_(RBS) in turnpositioned upstream of a third DNA sequence having at least onerestriction site that permits cleavage within six base pairs 3′ of theN_(RBS) sequence. Also useful is the phosphatase A (phoA) systemdescribed by Chang et al., in European Patent Publication No. 196,864,published Oct. 8, 1986. However, any available promoter systemcompatible with procaryotes can be used to construct a expression vectorof the invention.

In addition to bacteria, eucaryotic microbes, such as yeast, can also beused as recombinant host cells. Laboratory strains of Saccharomycescerevisiae, Baker's yeast, are most often used, although a number ofother strains are commonly available. While vectors employing the twomicron origin of replication are common, see Broach, 1983, Meth. Enz.101:307, other plasmid vectors suitable for yeast expression are known.See, e.g., Stinchcomb et al., 1979, Nature 282:39; Tschempe et al.,1980, Gene 10:157; and Clarke et al., 1983, Meth. Enz. 101:300. Controlsequences for yeast vectors include promoters for the synthesis ofglycolytic enzymes. See Hess et al., 1968, J. Adv. Enzyme Reg. 7:149;Holland et al., 1978, Biotechnology 17:4900; and Holland et al., 1981,J. Biol. Chem. 256:1385. Additional promoters known in the art includethe promoter for 3-phosphoglycerate kinase, see Hitzeman et al., 1980,J. Biol. Chem. 255:2073, and those for other glycolytic enzymes, such asglyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other promoters that have theadditional advantage of transcription controlled by growth conditionsare the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, and enzymes responsible for maltose and galactoseutilization (Holland, supra).

Terminator sequences may also be used to enhance expression when placedat the 3′ end of the coding sequence. Such terminators are found in the3′ untranslated region following the coding sequences in yeast-derivedgenes. Any vector containing a yeast-compatible promoter, origin ofreplication, and other control sequences is suitable for use inconstructing yeast expression vectors.

The coding sequence can also be expressed in eucaryotic host cellcultures derived from multicellular organisms. See, e.g., TissueCulture, Academic Press, Cruz and Patterson, editors (1973). Useful hostcell lines include COS-7, COS-A2, CV-1, murine cells such as murinemyelomas N51 and VERO, HeLa cells, and Chinese hamster ovary (CHO)cells. Expression vectors for such cells ordinarily include promotersand control sequences compatible with mammalian cells such as, forexample, the commonly used early and late promoters from Simian Virus 40(SV 40), see Fiers et al., 1978, Nature 273:113, or other viralpromoters such as those derived from polyoma, adenovirus 2, bovinepapilloma virus (BPV), or avian sarcoma viruses, or immunoglobulinpromoters and heat shock promoters. A system for expressing DNA inmammalian systems using a BPV vector system is disclosed in U.S. Pat.No. 4,419,446. A modification of this system is described in U.S. Pat.No. 4,601,978. General aspects of mammalian cell host systemtransformations have been described by Axel, U.S. Pat. No. 4,399,216.“Enhancer” regions are also important in optimizing expression; theseare, generally, sequences found upstream of the promoter region. Originsof replication may be obtained, if needed, from viral sources. However,integration into the chromosome is a common mechanism for DNAreplication in eucaryotes.

Plant cells can also be used as hosts, and control sequences compatiblewith plant cells, such as the nopaline synthase promoter andpolyadenylation signal sequences, see Depicker et al., 1982, J. Mol.Appl. Gen. 1:561, are available. Expression systems employing insectcells utilizing the control systems provided by baculovirus vectors havealso been described. See Miller et al., in Genetic Engineering (1986),Setlow et al., eds., Plenum Publishing, Vol. 8, pp. 277-97. Insectcell-based expression can be accomplished in Spodoptera frugipeida.These systems are also successful in producing recombinant enzymes.

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, 1972, Proc. Natl. Acad. Sci.USA 69:2110 is used for procaryotes or other cells that containsubstantial cell wall barriers. Infection with Agrobacteriumtumefaciens, see Shaw et al., 1983, Gene 23:315, is used for certainplant cells. For mammalian cells, the calcium phosphate precipitationmethod of Graham et al., 1978, Virology 52:546 is preferred.Transformations into yeast are carried out according to the method ofVan Solingen et al., 1977, J. Bact. 130:946, and Hsiao et al., 1979,Proc. Natl. Acad. Sci. USA 76:3829.

It may be desirable to modify the sequence of a DNA encoding apolypeptide comprising all or part of a reporter sequence, bindingsequence or reporter fusion of the invention to provide, for example, asequence more compatible with the codon usage of the host cell withoutmodifying the amino acid sequence of the encoded protein. Suchmodifications to the initial 5-6 codons may improve expressionefficiency. DNA sequences which have been modified to improve expressionefficiency, but which encode the same amino acid sequence, areconsidered to be equivalent and encompassed by the present invention.

A variety of site-specific primer-directed mutagenesis methods areavailable and well-known in the art. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989, secondedition, chapter 15.51, “Oligonucleotide-mediated mutagenesis,” which isincorporated herein by reference. The polymerase chain reaction (PCR)can be used to perform site-specific mutagenesis. In another techniquenow standard in the art, a synthetic oligonucleotide encoding thedesired mutation is used as a primer to direct synthesis of acomplementary nucleic acid sequence contained in a single-strandedvector, such as pBSM13+ derivatives, that serves as a template forconstruction of the extension product of the mutagenizing primer. Themutagenized DNA is transformed into a host bacterium, and cultures ofthe transformed bacteria are plated and identified. The identificationof modified vectors may involve transfer of the DNA of selectedtransformants to a nitrocellulose filter or other membrane and the“lifts” hybridized with kinased synthetic mutagenic primer at atemperature that permits hybridization of an exact match to the modifiedsequence but prevents hybridization with the original unmutagenizedstrand. Transformants that contain DNA that hybridizes with the probeare then cultured (the sequence of the DNA is generally confirmed bysequence analysis) and serve as a reservoir of the modified DNA.

Once the polypeptide has been expressed in a recombinant host cell,purification of the polypeptide may be desired. A variety ofpurification procedures can be used.

For long-term stability, the purified polypeptide can be stored in abuffer that contains one or more non-ionic polymeric detergents. Suchdetergents are generally those that have a molecular weight in the rangeof approximately 100 to 250,00 preferably about 4,000 to 200,000 daltonsand stabilize the enzyme at a pH of from about 3.5 to about 9.5,preferably from about 4 to 8.5. Examples of such detergents includethose specified on pages 295-298 of McCutcheon's Emulsifiers &Detergents, North American edition (1983), published by the McCutcheonDivision of MC Publishing Co., 175 Rock Road, Glen Rock, N.J. (USA), theentire disclosure of which is incorporated herein by reference.Preferably, the detergents are selected from the group comprisingethoxylated fatty alcohol ethers and lauryl ethers, ethoxylated alkylphenols, octylphenoxy polyethoxy ethanol compounds, modifiedoxyethylated and/or oxypropylated straight-chain alcohols, polyethyleneglycol monooleate compounds, polysorbate compounds, and phenolic fattyalcohol ethers. More particularly preferred are Tween 20™, apolyoxyethylated (20) sorbitan monolaurate from ICI Americas Inc.(Wilmington, Del.), and Iconol™ NP-40, an ethoxylated alkyl phenol(nonyl) from BASF Wyandotte Corp. (Parsippany, N.J.).

The following series of examples are presented by way of illustrationand not by way of limitation on the scope of the invention.

6. EXAMPLES

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Example 1 SGN17 His Scan Method

This example demonstrates that a binding sequence can be modified togenerate a binding sequence with a higher binding affinity and a bindingsequence with a pH dependent binding affinity.

pADEPT06 DNA Template:

A schematic of plasmid pADEPT06 is shown in FIG. 8. This plasmid is 5.2kb and encodes a single chain antibody variable region fragment (scFv)fused to β-lactamase (BLA) with a pelB leader sequence, and is driven bya lac promoter (P lac). The plasmid also carries a chloramphenicolresistance gene (CAT) as a selectable marker. This particular SGN17plasmid was made by a 3-piece ligation utilizing a linker. Two plasmidswere used to make pADEPT06: pCB04 for the vector fragment with the pel Bleader sequence, and pCR13 for the scFv-bla gene. pCBO4 was digestedwith HindIII and DraIII (both from New England Biolabs, Beverly, Mass.)resulting in a 2.7 kb fragment with the pCB04 backbone. pCR13 wasdigested with NdeI (Roche Molecular Biochemicals, Indianapolis, Ind.)and DraIII resulting in the 2.4 kb fragment containing the fusionprotein with the pelB leader sequence. Digests pCR13 were done in NEB2buffer from NEB (50 mM NaCl, 10 mN Tris-HCl, 10 mM MgCl₂, 1 mMdithiothreitol (pH 7.9 @ 25° C.). Both fragments were gel purified from1% agarose gel using a Qiagen kit (Qiagen, Valencia, Calif.). A linkersequence with 5′ HindIII complementary ends and 3′ NdeI complementaryends was used to link the 2.7 kb fragment and the 2.4 kb fragmentupstream of the pel B leader sequence. The pCB04 fragment was combinedwith the pCR13 fragment and the linker in a 1:1:10 molar ratio(respectively), using 17 μl DNA volume (95 ng total DNA) and 17 μlTakara ligase solution I (Panvera, Madison, Wis.) and incubatedovernight at 16° C. in a PTC-200™ machine (MJ Research, Waltham, Mass.).Sequencing information shows that the linker region is repeated upstreamof the leader sequence.

Mutagenic Primers:

Overlapping mutagenic primers were designed to replace certain aminoacids with histidine residues in the CDR3 regions of both the heavy andlight chains of the scFv portion of the scFv-BLA fusion. The wild-typecodon to be mutated was changed to the codon CAT (encoding histidine) ina pair of primers. The mutated codon in each primer was flanked on eachside by 17 nucleotides of wild-type sequence, unless the primer ended ina stretch of A residues; in this case, the flanking sequence wasextended so that it ended with a G or C residue. Each primer wasdesigned so that its mutant codon had the same number of nucleotidesflanking it on each side. Each primer was named according to themutation it was designed to create. For example, HCL100F is the forwardprimer for the heavy chain (HC) mutating the Leucine (L) in position100. Its overlapping primer is called HCL100R.

The names and sequences of the mutagenic oligos are provided in Table 1.TABLE 1 SGN17 His Scan Primers Heavy Chain HCK64F ACTACAATCCATCTCTC CATAGTCGCATTTCCATCAC HCK64R GTGATGGAAATGCGACTATGGAGAGATGGATTGTAGT HCR97FGCCACATATTACTGTGCA CAT AGGACTCTGGCTACTTAC HCR97RGTAAGTAGCCAGAGTCCTATGTGCACAGTAATATGTGGC HCR98F CATATTACTGTGCAAGA CATACTCTGGCTACTTACTA HCR98R TAGTAAGTAGCCAGAGTATGTCTTGCACAGTAATATG HCT99FATTACTGTGCAAGAAGG CAT CTGGCTACTTACTATGC HCT99RGCATAGTAAGTAGCCAGATGCCTTCTTGCACAGTAAT HCL100F ACTGTGCAAGAAGGACT CATGCTACTTACTATGCTAT HCL100R ATAGCATAGTAAGTAGCATGAGTCCTTCTTGCACAGT HCA101FGTGCAAGAAGGACTCTG CAT ACTTACTATGCTATGGA HCA101RTCCATAGCATAGTAAGTATGCAGAGTCCTTCTTGCAC HCT102F CAAGAAGGACTCTGGCT CATTACTATGCTATGGACTA HCT102R TAGTCCATAGCATAGTAATGAGCCAGAGTCCTTCTTG HCY103FGAAGGACTCTGGCTACT CAT TATGCTATGGACTACTG HCY103RCAGTAGTCCATAGCATAATGAGTAGCCAGAGTCCTTC HCY104F GGACTCTGGCTACTTAC CATGCTATGGACTACTGGGG HCY104R CCCCAGTAGTCCATAGCATGGTAAGTAGCCAGAGTCC HCA105FCTCTGGCTACTTACTAT CAT ATGGACTACTGGGGTCA HCA105RTGACCCCAGTAGTCCATATGATAGTAAGTAGCCAGAG HCM106F TGGCTACTTACTATGCT CATGACTACTGGGGTCAAGG HCM106R CCTTGACCCCAGTAGTCATGAGCATAGTAAGTAGCCA HCD107FCTACTTACTATGCTATG CAT TACTGGGGTCAAGGAAC HCD107RGTTCCTTGACCCCAGTAATGCATAGCATAGTAAGTAG HCY108F CTTACTATGCTATGGAC CATTGGGGTCAAGGAACCTC HCY108R GAGGTTCCTTGACCCCAATGGTCCATAGCATAGTAAG HCW109FACTATGCTATGGACTAC CAT GGTCAAGGAACCTCTGT HCW109RACAGAGGTTCCTTGACCATGGTAGTCCATAGCATAGT Light Chain LCR54FCAAAGCTCCTGATCTAC CAT GTTTCCAACCGATTTTC LCR54RGAAAATCGGTTGGAAACATGGTAGATCAGGAGCTTTG LCR58F GATTTTCTGGGGTCCCAGAC CATTTCAGTGGCAGTGGATCAGG LCR58R CCTGATCCACTGCCACTGAAATGGTCTGGGACCCCAGAAAATCLCQ94F GAGTTTATTTCTGCTCT CAT AGTACACATGTTCCTCC LCQ94RGGAGGAACATGTGTACTATGAGAGCAGAAATAAACTC LCS95F GTTTATTTCTGCTCTCAA CATACACATGTTCCTCCGACG LCS95R CGTCGGAGGAACATGTGTATGTTGAGAGCAGAAATAAAC LCT96FGTTTATTTCTGCTCTCAAAGT CAT CATGTTCCTCCGACGTTCGGT LCT96RACCGAACGTCGGAGGAACATGATGACTTTGAGAGCAGAAATAAAC LCH97F TCTGCTCTCAAAGTACACAT GTTCCTCCGACGTTCGG LCH97R CCGAACGTCGGAGGAACATGTGTACTTTGAGAGCAGALCV98F GCTCTCAAAGTACACAT CAT CCTCCGACGTTCGGTGG LCV98RCCACCGAACGTCGGAGGATGATGTGTACTTTGAGAGC LCP99F CTCAAAGTACACATGTT CATCCGACGTTCGGTGGAGG LCP99R CCTCCACCGAACGTCGGATGAACATGTGTACTTTGAG LCP100FCAAAGTACACATGTTCCT CAT ACGTTCGGTGGAGGCACC LCP100RGGTGCCTCCACCGAACGTATGAGGAACATGTGTACTTTG LCT101F AGTACACATGTTCCTCCG CATTTCGGTGGAGGCACCAAG LCT101R CTTGGTGCCTCCACCGAAATGCGGAGGAACATGTGTACTAll sequences written 5′-3′. Mutagenic codon in bold and underlined.

A QUICKCHANGE™ site directed mutagenesis kit (Stratagene, La Jolla,Calif.) was used to set up PCR amplifications as follows: H₂O 39 μl 10xbuffer 5 μl dNTP mix 1.5 μl Forward primer 1 μl (0.5 μM finalconcentration) Reverse primer 1 μl (0.5 μM final concentration) pfupolymerase 1 μl Plasmid DNA 1.5 μl (150 ng) Total 50 μl

The buffer comprised 100 mM KCl, 100 mM (NH₄)SO₄, 200 mM Tris-HCl (pH8.8), 20 mM MgSO₄, 1% Triton X-100, 1 mg/ml nuclease-free bovine serumalbumin (BSA).

The following touchdown PCR program was used in a PTC-200™ machine (MJResearch, Waltham, Mass.):

-   -   1) 95° C., 2 minutes    -   2) 95° C., 45 seconds    -   3) 60° C., 1 minute (Reduced by 1.0° C. per cycle)    -   4) 68° C. 11 minutes (i.e., 2 minutes per kb, 5 kb plasmid, plus        an additional minute)    -   5) Go to step (2) for 9 cycles    -   6) 95° C., 45 seconds    -   7) 50° C., 1 minute    -   8) 68° C., 11 minutes    -   9) Go to step (6) for 5 cycles    -   10) Hold at 4° C.

A negative control without primers was also set up and carried throughall steps.

DpnI Digest:

DpnI is a restriction enzyme that cuts methylated and hemimethylated,but not unmethylated, double-stranded DNA. After PCR, 1 μl of DpnI wasadded to each reaction to digest template DNA, which is methylated, butnot amplified DNA, most of which is unmethylated, thus reducing thebackground of wild-type sequence. A sample of the control was savedbefore digestion. Digests were incubated at 37° C. for 1.5 hrs, theneach reaction was spiked with an additional 1 μl of DpnI and incubatedanother 1.5 hrs. Reactions were run on a gel after digests alongside thecontrol amplification before and after DpnI digestion. All reactionsappeared to work, and, as expected, the control band was fully digestedby DpnI.

Transformation:

1 μl of each reaction (not purified), including the digested control,were used to transform 50 μl of Top 10 electro-competent cells(Invitrogen, Carlsbad, Calif.) and 250 μl SOC medium (2% Bacto-Tryptone,0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl) was added. The cellswere shaken at 37° C. for 45 min, then 30 μl of a 1 to 10 dilution wasplated (i.e., one tenth of the total volume of each transformation wasplated) on both 5 ppm chloramphenicol (CMP) and 5 ppm CMP+0.1 ppmcefotaxime (CTX) plates. Plates were incubated overnight at 37° C.Transformation results are provided in Table 2. TABLE 2 CMP CMP + CTX %ACTIVE (control) 0 0 0 ME43 14 5 36 ME44 120 34 28 ME45 784 236 32 ME46440 159 36 ME47 516 184 36 ME48 268 62 23 ME49 30 10 33 ME50 488 61 12.5ME51 316 57 18 ME52 380 192 50 ME53 440 80 18 ME54 968 308 32 ME55 356148 42 ME56 90 17 19 ME57 424 112 26 ME58 38 10 26 ME59 141 53 38 ME60212 144 68 ME61 90 27 30 ME62 268 87 32 (WT codon) ME63 296 88 30 ME64196 112 57 ME65 168 128 76 ME66 236 76 32

All bacteria transformed by and expressing a plasmid produced colonieson the CTX plate, and thus provided a measure of the efficiency oftransformation. However, only bacteria transformed by plasmidscontaining a functional BLA grew on the CTX+CMP plates.

Clone names in Table 2 are listed in the same order as the primer pairsused to make them are listed in Table 1, e.g., ME43 was created usingprimer pair HCK64F/R, ME44 was created using primer pair HCR97F/R, andso on.

Four colonies were picked for each transformation (excluding LCH97because this represents the wild-type sequence; pADEPT06 WT colonieswere picked as a control). Picked colonies were first swirled into a 96well plate with membrane bottom, each well containing 200 ul LB+5 ppmCMP, and then put into the corresponding well of another 96 well platewithout filter, to be used as a stock plate.

The 96 well plates were incubated at 25° C. in a humidified box withshaking for 48 hrs. Glycerol was added to the stock plate to a finalconcentration of 10% and stored at −80° C.

Screening Mutants:

Target protein p97 (prepared, for example, by the method set forth inSiemers, N. O., D. E. Kerr, S. Yarnold, M. R. Stebbins, V. M. Vrudhula,I. Hellstrom and P. D. Senter (1997), Bioconj Chem 8, 510-519.Construction, expression and activities of L49-sFv-beta-lactamase, asingle-chain antibody fusion protein for anticancer prodrug activation)was immobilized on a polystyrene plate by adding 100 μl of 1 μg/ml p97in PBS and incubating the plate at 4° C. overnight. The plate is thenwashed with PBST (PBS+0.25% Tween 20) and blocked with 200 μl/well of 1%casein in PBS overnight at 4° C. On the day of screening, the plate waswashed with PBST, then each well received 80 μl of 50 mM PBS pH7.4 and20 μl of cell culture broth from each mutant. The plate was incubated atroom temperature with gentle shaking to let SGN-17 bind to immobilizedp97 on the plate. The amount of each mutant enzyme bound to p97 wasdetermined at two time points. After 1 hour, the plate was washed withPBST, and 200 μl of the BLA substrate nitrocefin in 50 mM PBS bufferpH7.4 or pH6.5 was added into each well. The amount of bound SGN-17 wasmeasured by monitoring hydrolysis of nitrocefin at wavelength 490 nm.This was the T₀ time point measurement. The plate was then incubated ineach substrate buffer for one hour, providing an opportunity for boundmutant SGN-17 to dissociate, then quickly rinsed with PBST. Theremaining bound SGN-17 was measured by again monitoring the hydrolysisof substrate nitrocefin in each buffer. This was the T₁ time pointmeasurement. A ratio of bound activity at T₁ vs. T₀ was calculated foreach mutant, and an index was calculated by dividing the ratio of mutantover parent, as shown in Table 3. TABLE 3 Mutants sequence positionregion Index pH 7.4 Index pH 6.5 ME43 K HC62 CDR2 0.61 0.65 ME44 R HC94CDR3 0.24 0 ME45 R HC95 CDR3 0 0 ME46 T HC96 CDR3 0.38 0.09 ME47 L HC97CDR3 0.24 0 ME48 A HC98 CDR3 0.49 0.33 ME50 Y HC100 CDR3 0.33 0 ME51 YHC101 CDR3 0.26 0 ME52 A HC102 CDR3 0 0 ME53 M HC103 CDR3 0.97 0.8 ME54D HC104 CDR3 0.41 0.7 ME55 Y HC105 CDR3 0.8 0.7 ME56 W HC106 CDR3 0.570.41 ME58 R LC58 CDR2 0.92 0.76 ME59 Q LC94 CDR3 0.28 0 ME60 S LC95 CDR31.04 1.09 ME61 T LC96 CDR3 0.82 0.81 ME63 V LC98 CDR3 0.21 0 ME64 P LC99CDR3 0.35 0 ME65 P LC100 CDR3 0 0 ME66 T LC101 CDR3 1.36 1.73

A high index value for a mutant indicates that it has a slow k_(off). Anindex value of 0 indicates that no binding was detected for the mutantat that pH.

These data illustrate that many residues in the CDR3 of SGN-17 can bereplaced with His while retaining various degrees of binding affinity.Mutagenesis at position LC101 actually leads to an increase in bindingaffinity which is larger at pH 6.5 as compared to pH 7.4. Thus, theintroduction of a His in position LC101 affects the pH-dependence oftarget binding of SGN-17. Comparing the index values at both pH valuesshows that several of the tested mutations affect pH-dependence ofbinding. Stronger effects can be achieved by adding further mutations,by testing substitutions other then His, by testing substitutions,insertions or deletions at more positions of the binding moiety, or byextending the mutagenesis to the BLA part of the fusion protein.

Example 2 Affinity Maturation of an scFv by Site Saturation ScanningMutagenesis

A. Generation of Site Saturation Libraries

64 site saturation mutagenesis libraries were generated. In each ofthese libraries, one codon, that codes for a CDR position (as defined bythe Kabat nomenclature) in ME66.4-scFv, exactly the same as ME66, wasrandomized. The libraries were generated using the QuikChange protocol(Stratagene, La Jolla, Calif.) essentially as recommended by themanufacturer. Each reaction used two mutagenic oligonucleotides whichhad the following design: 17 perfect matches flanking the random codonon each side, NNS in place of the random codon. For example, libraryME67 used the forward primer CTGGCGACTCCATCACCNNSGGTTACTGGAACTGGAT andthe reverse primer ATCCAGTTCCAGTAACCSNNGGTGATGGAGTCGCCAG, where Nrepresents a mixture of A, T, G, and C and S represents a mixture of Gand C. This approach allows for the generation of 32 different codonswhich encode all 20 amino acids. After the QuikChange reaction and Dpn Idigest, which degrades parent plasmid, the reaction mixture was used totransform TOP10 cells (Invitrogen, Carlsbad, Calif.) by electroporation.TABLE 4 oligonucleotides used to generate the 64 site saturationlibraries: Heavy Chain H31 CTGGCGACTCCATCACCNNSGGTTACTGGAACTGGAT H31ATCCAGTTCCAGTAACCSNNGGTGATGGAGTCGCCAG H32GCGACTCCATCACCAGTNNSTACTGGAACTGGATCCG H32CGGATCCAGTTCCAGTASNNACTGGTGATGGAGTCGC H33ACTCCATCACCAGTGGTNNSTGGAACTGGATCCGGCA H33TGCCGGATCCAGTTCCASNNACCACTGGTGATGGAGT H34TCCATCACCAGTGGTTACNNSAACTGGATCCGGCAGTTC H34GAACTGCCGGATCCAGTTSNNGTAACCACTGGTGATGGA H50AACTTGAATATATGGGTNNSATAAGCGACAGTGGTAT H50ATACCACTGTCGCTTATSNNACCCATATATTCAAGTT H51TTGAATATATGGGTTACNNSAGCGACAGTGGTATCAC H51GTGATACCACTGTCGCTSNNGTAACCCATATATTCAA H52GAATATATGGGTTACATANNSGACAGTGGTATCACTTAC H52GTAAGTGATACCACTGTCSNNTATGTAACCCATATATTC H53TATATGGGTTACATAAGCNNSAGTGGTATCACTTACTAC H53GTAGTAAGTGATACCACTSNNGCTTATGTAACCCATATA H54ATGGGTTACATAAGCGACNNSGGTATCACTTACTACAAT H54ATTGTAGTAAGTGATACCSNNGTCGCTTATGTAACCCAT H55GTTACATAAGCGACAGTNNSATCACTTACTACAATCC H55GGATTGTAGTAAGTGATSNNACTGTCGCTTATGTAAC H56ACATAAGCGACAGTGGTNNSACTTACTACAATCCATC H56GATGGATTGTAGTAAGTSNNACCACTGTCGCTTATGT H57TAAGCGACAGTGGTATCNNSTACTACAATCCATCTCT H57AGAGATGGATTGTAGTASNNGATACCACTGTCGCTTA H58TAAGCGACAGTGGTATCACTNNSTACAATCCATCTCTCAAAAG H58CTTTTGAGAGATGGATTGTASNNAGTGATACCACTGTCGCTTA H59GACAGTGGTATCACTTACNNSAATCCATCTCTCAAAAGT H59ACTTTTGAGAGATGGATTSNNGTAAGTGATACCACTGTC H60GTGGTATCACTTACTACNNSCCATCTCTCAAAAGTCG H60CGACTTTTGAGAGATGGSNNGTAGTAAGTGATACCAC H61GTATCACTTACTACAATNNSTCTCTCAAAAGTCGCAT H61ATGCGACTTTTGAGAGASNNATTGTAGTAAGTGATAC H62TCACTTACTACAATCCANNSCTCAAAAGTCGCATTTC H62GAAATGCGACTTTTGAGSNNTGGATTGTAGTAAGTGA H63CTTACTACAATCCATCTNNSAAAAGTCGCATTTCCAT H63ATGGAAATGCGACTTTTSNNAGATGGATTGTAGTAAG H64ACTACAATCCATCTCTCNNSAGTCGCATTTCCATCAC H64GTGATGGAAATGCGACTSNNGAGAGATGGATTGTAGT H65ACAATCCATCTCTCAAANNSCGCATTTCCATCACTCG H65CGAGTGATGGAAATGCGSNNTTTGAGAGATGGATTGT H97GCCACATATTACTGTGCANNSAGGACTCTGGCTACTTAC H97GTAAGTAGCCAGAGTCCTSNNTGCACAGTAATATGTGGC H98CATATTACTGTGCAAGANNSACTCTGGCTACTTACTA H98TAGTAAGTAGCCAGAGTSNNTCTTGCACAGTAATATG H99ATTACTGTGCAAGAAGGNNSCTGGCTACTTACTATGC H99GCATAGTAAGTAGCCAGSNNCCTTCTTGCACAGTAAT H100ACTGTGCAAGAAGGACTNNSGCTACTTACTATGCTAT H100ATAGCATAGTAAGTAGCSNNAGTCCTTCTTGCACAGT H101GTGCAAGAAGGACTCTGNNSACTTACTATGCTATGGA H101TCCATAGCATAGTAAGTSNNCAGAGTCCTTCTTGCAC H102CAAGAAGGACTCTGGCTNNSTACTATGCTATGGACTA H102TAGTCCATAGCATAGTASNNAGCCAGAGTCCTTCTTG H103GAAGGACTCTGGCTACTNNSTATGCTATGGACTACTG H103CAGTAGTCCATAGCATASNNAGTAGCCAGAGTCCTTC H104GGACTCTGGCTACTTACNNSGCTATGGACTACTGGGG H104CCCCAGTAGTCCATAGCSNNGTAAGTAGCCAGAGTCC H105CTCTGGCTACTTACTATNNSATGGACTACTGGGGTCA H105TGACCCCAGTAGTCCATSNNATAGTAAGTAGCCAGAG H106TGGCTACTTACTATGCTNNSGACTACTGGGGTCAAGG H106CCTTGACCCCAGTAGTCSNNAGCATAGTAAGTAGCCA H107CTACTTACTATGCTATGNNSTACTGGGGTCAAGGAAC H107GTTCCTTGACCCCAGTASNNCATAGCATAGTAAGTAG H108CTTACTATGCTATGGACNNSTGGGGTCAAGGAACCTC H108GAGGTTCCTTGACCCCASNNGTCCATAGCATAGTAAG H109ACTATGCTATGGACTACNNSGGTCAAGGAACCTCTGT H109ACAGAGGTTCCTTGACCSNNGTAGTCCATAGCATAGT Light Chain L24CCTCCATCTCTTGCAGGNNSAGTCAGAGCCTTGTACA L24TGTACAAGGCTCTGACTSNNCCTGCAAGAGATGGAGG L25CCATCTCTTGCAGGGCTNNSCAGAGCCTTGTACACAG L25CTGTGTACAAGGCTCTGSNNAGCCCTGCAAGAGATGG L26ATCTCTTGCAGGGCTAGTNNSAGCCTTGTACACAGTAAT L26ATTACTGTGTACAAGGCTSNNACTAGCCCTGCAAGAGAT L27CTTGCAGGGCTAGTCAGNNSCTTGTACACAGTAATGG L27CCATTACTGTGTACAAGSNNCTGACTAGCCCTGCAAG L28TGCAGGGCTAGTCAGAGCNNSGTACACAGTAATGGAAAC L28GTTTCCATTACTGTGTACSNNGCTCTGACTAGCCCTGCA L29GGGCTAGTCAGAGCCTTNNSCACAGTAATGGAAACAC L29GTGTTTCCATTACTGTGSNNAAGGCTCTGACTAGCCC L30CTAGTCAGAGCCTTGTANNSAGTAATGGAAACACCTA L30TAGGTGTTTCCATTACTSNNTACAAGGCTCTGACTAG L31TAGTCAGAGCCTTGTACACNNSAATGGAAACACCTATTTAC L31GTAAATAGGTGTTTCCATTSNNGTGTACAAGGCTCTGACTA L32AGAGCCTTGTACACAGTNNSGGAAACACCTATTTACA L32TGTAAATAGGTGTTTCCSNNACTGTGTACAAGGCTCT L33GCCTTGTACACAGTAATNNSAACACCTATTTACATTG L33CAATGTAAATAGGTGTTSNNATTACTGTGTACAAGGC L34TTGTACACAGTAATGGANNSACCTATTTACATTGGTA L34TACCAATGTAAATAGGTSNNTCCATTACTGTGTACAA L35TACACAGTAATGGAAACNNSTATTTACATTGGTACC L35GGTACCAATGTAAATASNNGTTTCCATTACTGTGTA L36ACAGTAATGGAAACACCNNSTTACATTGGTACCTGCA L36TGCAGGTACCAATGTAASNNGGTGTTTCCATTACTGT L37AGTAATGGAAACACCTATNNSCATTGGTACCTGCAGAAG L37CTTCTGCAGGTACCAATGSNNATAGGTGTTTCCATTACT L38ATGGAAACACCTATTTANNSTGGTACCTGCAGAAGCC L38GGCTTCTGCAGGTACCASNNTAAATAGGTGTTTCCAT L53CTCCAAAGCTCCTGATCNNSAGAGTTTCCAACCGATT L53AATCGGTTGGAAACTCTSNNGATCAGGAGCTTTGGAG L54CAAAGCTCCTGATCTACNNSGTTTCCAACCGATTTTC L54GAAAATCGGTTGGAAACSNNGTAGATCAGGAGCTTTG L55AGCTCCTGATCTACAGANNSTCCAACCGATTTTCTGG L55CCAGAAAATCGGTTGGASNNTCTGTAGATCAGGAGCT L56TCCTGATCTACAGAGTTNNSAACCGATTTTCTGGGGT L56ACCCCAGAAAATCGGTTSNNAACTCTGTAGATCAGGA L57TGATCTACAGAGTTTCCNNSCGATTTTCTGGGGTCCC L57GGGACCCCAGAAAATCGSNNGGAAACTCTGTAGATCA L58TCTACAGAGTTTCCAACNNSTTTTCTGGGGTCCCAGA L58TCTGGGACCCCAGAAAASNNGTTGGAAACTCTGTAGA L59ACAGAGTTTCCAACCGANNSTCTGGGGTCCCAGACAG L59CTGTCTGGGACCCCAGASNNTCGGTTGGAAACTCTGT L60GAGTTTCCAACCGATTTNNSGGGGTCCCAGACAGGTT L60AACCTGTCTGGGACCCCSNNAAATCGGTTGGAAACTC L94GAGTTTATTTCTGCTCTNNSAGTACACATGTTCCTCC L94GGAGGAACATGTGTACTSNNAGAGCAGAAATAAACTC L95GAGTTTATTTCTGCTCTCAANNSACACATGTTCCTCCGCATTT L95AAATGCGGAGGAACATGTGTSNNTTGAGAGCAGAAATAAACTC L96TATTTCTGCTCTCAAAGTNNSCATGTTCCTCCGCATTTC L96GAAATGCGGAGGAACATGSNNACTTTGAGAGCAGAAATA L97TCTGCTCTCAAAGTACANNSGTTCCTCCGCATTTCGG L97CCGAAATGCGGAGGAACSNNTGTACTTTGAGAGCAGA L98GCTCTCAAAGTACACATNNSCCTCCGCATTTCGGTGG L98CCACCGAAATGCGGAGGSNNATGTGTACTTTGAGAGC L99CTCAAAGTACACATGTTNNSCCGCATTTCGGTGGAGG L99CCTCCACCGAAATGCGGSNNAACATGTGTACTTTGAG L100CAAAGTACACATGTTCCTNNSCATTTCGGTGGAGGCACC L100GGTGCCTCCACCGAAATGSNNAGGAACATGTGTACTTTG L101AGTACACATGTTCCTCCGNNSTTCGGTGGAGGCACCAAG L101CTTGGTGCCTCCACCGAASNNCGGAGGAACATGTGTACT

B. Screen for Improved Binding

Libraries were plated onto agar plates containing LB medium and 5 mg/lchloramphenicol and 0.1 mg/l cefotaxime (Sigma). 88 colonies from eachlibrary and parent colonies were picked and inoculated into 384-wellplates containing 80 ul LB containing 5 mg/l chloramphenicol and 0.1mg/l cefotaxime. Plates were incubated at 25 C in humidified boxes withshaking for 48 hrs.

Target protein p97 was immobilized in 384-well polystyrene plates byadding 40 ul of 1 ug/ml p97 in PBS and incubating the plate at 4 Covernight. The plates were then washed with PBST (PBS+0.1% Tween-20) andblocked with 200 ul/well of 1% Casein in PBS overnight at 4 C. On theday of screening, the plates were washed with PBST. Subsequently, 24ul/well of 50 mM PBS pH7.4 was first added into plate each well followedby 8 ul of cell culture broth from expression plates. The plate wasincubated at room temperature with gentle shaking to let ME66-scFv tobind to immobilized P97 on the plate. After 1 hour, the plate was washedwith PBST and 200 ul of BLA assay buffer containing 0.1 mg/ml nitrocefin(Oxoid, New York) in 50 mM PBS buffer pH6.5 was added into each well,the bound ME66scFv was measured by monitoring hydrolysis of nitrocefinat wavelength 490 nm. The plate was then left incubated in substratebuffer to allow the bound ME66scFv-BLA to dissociate, after 1.5 hour theplate was quickly rinsed with PBST. The remaining bound ME66scFv-BLA wasagain measured by monitoring the hydrolysis of freshly added substratenitrocefin. Dissociation of ME66-scFv from p97 was monitored again after3-5 hours. A ratio of bound activity at time 1 vs. time 0 or time 2 vs.time 0 was calculated for each mutant from dissociation data, an indexat each time point was further calculated by dividing ratio of mutantover parent, and winner mutants were chosen if they had a high index.

After the primary screening, 21 winners were chosen for repeat analysisin quadruplicates. Each winner was streaked out on LA agar containing 5mg/l chloramphenicol, 4 colonies from each winner were transferred in 96well plate containing 200 ul/well LB containing 5 mg/l chloramphenicol.Some wells were inoculated with ME66.4 as a reference. The plate wasincubated at 25 C for 70 hours. Target protein p97 was bitotinylated andimmobilized in 96 well neutravidin (Pierce, Rockford, Ill.) plate at ap97 concentration of 5 ug/ml of 100 ul/well, the plate was then blockedwith 1% Casein. On the day of screening, 70 ul/well of PBS buffer pH7.4was added into target plate, and 20 ul/well of culture broth wastransferred from expression plate to target plate. The plates wereincubated at room temperature for 1 hour, and were then washed withPBST. 200 ul of BLA substrate nitrocefin in 50 mM PBS buffer pH6.5 wereadded into each well, and the bound ME66scFv was measured by monitoringhydrolysis of nitrocefin at wavelength 490 nm. The plate was leftincubated in substrate buffer for an additional 1.5 hour. After quickrinsing with PBST, the bound ME66scFv-BLA was again measured usingnitrocefin. The dissociation of ME66scFv from p97 was again measuredbetween 3-6 hours after the initial time point and a binding index wascalculated at 2 time points. In parallel, the plate was screened underidentical conditions but using 50 mM PBS buffer at pH 7.4. Data werenormalized as described in Example 1. The normalized screening resultsmeasured at pH 6.5 and at pH 7.4 are shown in the FIG. 9.

Table 6, below, shows mutations that have been observed in the threebest variants. TABLE 4 Mutations in affinity matured variants Clonemutation ME70.1 heavy chain, S65K ME70.7 heavy chain, S65P ME81.3 heavychain, N60R

Example 3 Stabilization of an scFv

A. Construction of pME27.1

Plasmid pME27.1 was generated by inserting a Bgl I EcoRV fragmentencoding a part of the pelB leader, the CAB1-scFv and a small part ofBLA into plasmid the expression vector pME25. The insert, encoding forthe CAB1-scFv, has been synthesized by Aptagen (Herndon, Va.) based onthe sequence of the scFv MFE-23 that was described in [Boehm, M. K., A.L. Corper, T. Wan, M. K. Sohi, B. J. Sutton, J. D. Thornton, P. A. Keep,K. A. Chester, R. H. Begent and S. J. Perkins (2000) Biochem J 346 Pt 2,519-28, Crystal structure of the anti-(carcinoembryonic antigen)single-chain Fv antibody MFE-23 and a model for antigen binding based onintermolecular contacts]. Both the plasmid containing the synthetic gene(pPCR-GME1) and pME25 were digested with BglI and EcoRV, gel purifiedand ligated together with Takara ligase. Ligation was transformed intoTOP10 (Invitrogen, Carlsbad, Calif.) electrocompetent cells, plated onLA medium containing 5 mg/l chloramphenicol and 0.1 mg/l cefotaxime.

Plasmid pME27.1 contains the following features:

-   P lac: 4992-5113 bp-   pel B leader: 13-78-   CAB 1 scFv: 79-810-   BLA: 811-1896-   T7 term.: 2076-2122-   CAT: 3253-3912

A schematic of plasmid pME27.1 can be found in FIG. 10A. The CAB1sequence, indicating heavy and light chain domains, can be found in FIG.10B; the amino acid sequence can also be found in 10D, with linker andBLA.

B. Choosing Mutations for Mutagenesis

The sequence of the vH and vL sequences of CAB1-scFv were compared witha published frequency analysis of human antibodies (Boris Steipe (1998)Sequenzdatenanalyse. (“Sequence Data Analysis”, available in Germanonly) in Bioanalytik eds. H. Zorbas und F. Lottspeich, SpektrumAkademischer Verlag. S. 233-241). The authors aligned sequences ofvariable segments of human antibodies as found in the Kabat data baseand calculated the frequency of occurrence of each amino acid for eachposition. These alignment can be seen in FIG. 12. Specifically, FIG. 12Ashows an alignment of the observed frequencies of the five most abundantamino acids in alignment of human sequences in the heavy chain. FIG. 12Bshows an alignment of the observed frequencies of the five most abundantamino acids in alignment of human sequences in the light chain.

We compared these frequencies with the actual amino acid sequence ofCAB1 and identified 33 positions that fulfilled the following criteria:

-   -   The position is not part of a CDR as defined by the Kabat        nomenclature.    -   The amino acid found in CAB1-scFv is observed in the homologous        position in less than 10% of human antibodies    -   The position is not one of the last 6 amino acids in the light        chain of scFv.        The resulting 33 positions were chosen for combinatorial        mutagenesis. Mutagenic oligonucleotides were synthesized for        each of the 33 positions such that the targeted position would        be changed from the amino acid in CAB1-scFv to the most abundant        amino acid in the homologous position of a human antibody. FIG.        10B shows the sequence of CAB1-scFv, the CDRs, and the mutations        that were chosen for combinatorial mutagenesis.

C. Construction of Library NA05

Table 5 listing the sequences of 33 mutagenic oligonucleotides that wereused to generate combinatorial library NA05: TABLE 5 pos. (pME27) countresidues to MFE-23 (VH) be changed QuikChange multi primer  3 K Qnsa147.1fp CGGCCATGGCCCAGGTGCAGCTGCAGCAGTCTGGGGC  13 R K nsa147.2fpCTGGGGCAGAACTTGTGAAATCAGGGACCTCAGTCAA  14 S P nsa147.3fpGGGCAGAACTTGTGAGGCCGGGGACCTCAGTCAAGTT  16 T G nsa147.4fpAACTTGTGAGGTCAGGGGGCTCAGTCAAGTTGTCCTG  28 N T nsa147.5fpGCACAGCTTCTGGCTTCACCATTAAAGACTCCTATAT  29 I F nsa147.6fpCAGCTTCTGGCTTCAACTTTAAAGACTCCTATATGCA  30 K S nsa147.7fpCTTCTGGCTTCAACATTAGCGACTCCTATATGCACTG  37 L V nsa147.8fpACTCCTATATGCACTGGGTGAGGCAGGGGCCTGAACA  40 G A nsa147.9fpTGCACTGGTTGAGGCAGGCGCCTGAACAGGGCCTGGA  42 E G nsa147.10fpGGTTGAGGCAGGGGCCTGGCCAGGGCCTGGAGTGGAT  67 K R nsa147.11fpCCCCGAAGTTCCAGGGCCGTGCCACTTTTACTACAGA  68 A F nsa147.12fpCGAAGTTCCAGGGCAAGTTCACTTTTACTACAGACAC  70 F I nsa147.13fpTCCAGGGCAAGGCCACTATTACTACAGACACATCCTC  72 T R nsa147.14fpGCAAGGCCACTTTTACTCGCGACACATCCTCCAACAC  76 S K nsa147.15fpTTACTACAGACACATCCAAAAACACAGCCTACCTGCA  97 N A nsa147.16fpCTGCCGTCTATTATTGTGCGGAGGGGACTCCGACTGG  98 E R nsa147.17fpCCGTCTATTATTGTAATCGCGGGACTCCGACTGGGCC 136 E Q nsa147.18fpCTGGCGGTGGCGGATCACAGAATGTGCTCACCCAGTC 137 N S nsa147.19fpGCGGTGGCGGATCAGAAAGCGTGCTCACCCAGTCTCC 142 S P nsa147.20fpGAAAATGTGCTCACCCAGCCGCCAGCAATCATGTCTGC 144 A S nsa147.21fpTGCTCACCCAGTCTCCAAGCATCATGTCTGCATCTCC 146 M V nsa147.22fpCCCAGTCTCCAGCAATCGTGTCTGCATCTCCAGGGGA 152 E Q nsa147.23fpTGTCTGCATCTCCAGGGCAGAAGGTCACCATAACCTG 153 K T nsa147.24fpCTGCATCTCCAGGGGAGACCGTCACCATAACCTGCAG 170 F Y nsa147.25fpTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGCAC 181 W V nsa147.26fpGCACTTCTCCCAAACTCGTGATTTATAGCACATCCAA 194 A D nsa147.27fpTGGCTTCTGGAGTCCCTGATCGCTTCAGTGGCAGTGG 200 G K nsa147.28fpCTCGCTTCAGTGGCAGTAAATCTGGGACCTCTTACTC 205 Y A nsa147.29fpGTGGATCTGGGACCTCTGCGTCTCTCACAATCAGCCG 212 M L nsa147.30fpCTCTCACAATCAGCCGACTGGAGGCTGAAGATGCTGC 217 A E nsa147.31fpGAATGGAGGCTGAAGATGAAGCCACTTATTACTGCCA 219 T D nsa147.32fpAGGCTGAAGATGCTGCCGATTATTACTGCCAGCAAAG 234 A G nsa147.33fpACCCACTCACGTTCGGTGGCGGCACCAAGCTGGAGCT

The QuikChange multi site-directed mutagenesis kit (QCMS; StratageneCatalog # 200514) was used to construct the combinatorial library NA05using 33 mutagenic primers. The primers were designed so that they had17 bases flanking each side of the codon of interest based on thetemplate plasmid pME27.1. The codon of interest was changed to encodethe appropriate consensus amino acid using an E.coli codon usage table.All primers were designed to anneal to the same strand of the templateDNA (i.e., all were forward primers in this case). The QCMS reaction wascarried out as described in the QCMS manual with the exception of theprimer concentration used, which ecommends using 50 ng of each primer inthe reaction whereas we used around 3 ng of each primer. Other primeramounts may be used. In particular, the reaction contained 50-100 ngtemplate plasmid (pME27.1; 5178 bp), 1 μl of primers mix (10 μM stock ofall primers combined containing 0.3 μM each primer), 1 μl dNTPs (QCMSkit), 2.5 μl 10× QCMS reaction buffer, 18.5 μl deoinized water, and 1 μlenzyme blend (QCMS kit), for a total volume of 25 μl. The thermocyclingprogram was 1 cycle at 95° for 1 min., followed by 30 cycles of 95° C.for 1 min., 55° C. for 1 min., and 65° C. for 10 minutes. DpnI digestionwas performed by adding 1 μl DpnI (provided in the QCMS kit), incubationat 37° C. for 2 hours, addition of another 1 μl DpnI, and incubation at37° C. for an additional 2 hours. 1 μl of the reaction was transformedinto 50 μl of TOP10 electrocompetent cells from Invitrogen. 250 μl ofSOC was added after electroporation, followed by a 1 hr incubation withshaking at 37° C. Thereafter, 10-50 μl of the tranformation mix wasplated on LA plates with 5 ppm chloramphenicol (CMP) or LA plates with 5ppm CMP and 0.1 ppm of cefotaxime (CTX) for selection of active BLAclones. The active BLA clones from the CMP+CTX plates were used forscreening, whereas the random library clones from the CMP plates weresequenced to assess the quality of the library.

16 randomly chosen clones were sequenced. The clones contained differentcombinations of 1 to 7 mutations.

D. Screen for Improved Expression

We found that when TOP10/pME27.1 is cultured in LB medium at 37 C thenthe concentration of intact fusion protein peaks after one day and mostof the fusion protein is degraded by host proteases after 3 days ofculture. Degradation seems to occur mainly in the scFv portion of theCAB1 fusion protein as the cultures contain significant amounts of freeBLA after 3 days, which can be detected by Western blotting, ornitrocefin (Oxoid, New York) activity assay. Thus we applied a screen tolibrary NA05 that was able to detect variants of CAB1-scFv that wouldresist degradation by host proteases over 3 days of culture at 37 C.

Library NA05 was plated onto agar plates with LA medium containing 5mg/l chloramphenicol and 0.1 mg/l cefotaxime (Sigma). 910 colonies weretransferred into a total of 10 96-well plates containing 100 ul/well ofLA medium containing 5 mg/l chloramphenicol and 0.1 mg/l cefotaxime.Four wells in each plate were inoculated with TOP10/pME27.1 as controland one well per plate was left as a blank. The plates were grownovernight at 37 C. The next day the cultures were used to inoculatefresh plates (production plates) containing 100 ul of the same mediumusing a transfer stamping tool and glycerol was added to the masterplates which were stored at −70 C. The production plates were incubatedin a humidified shaker at 37 C for 3 days. 100 ul of BPER (Pierce,Rockford, Ill.) per well was added to the production plate to releaseprotein from the cells. The production plate was diluted 100-fold inPBST (PBS containing 0.125% Tween-20) and BLA activity was measured bytransferring 20 ul diluted lysate into 180 ul of nitrocephin assaybuffer (0.1 mg/ml nitrocephin in 50 mM PBS buffer containing 0.125%octylglucopyranoside (Sigma)) and the BLA activity was determined at 490nm using a Spectramax plus plate reader (Molecular Devices, Sunnyvale,Calif.).

Binding to CEA (carcinoembryonic antigen, Biodesign Intl., Saco, Me.)was measured using the following procedure: 96-well plates were coatedwith 100 ul per well of 5 ug/ml of CEA in 50 mM carbonate buffer pH 9.6overnight. The plates were washed with PBST and blocked for 1-2 hourswith 300 ul of casein (Pierce, Rockford, Ill.). 100 ul of sample fromthe production plate diluted 100-1000 fold was added to the CEA coatedplate and the plates were incubated for 2 h at room temperature.Subsequently, the plates were washed four times with PBST and 200 ulnitrocefin assay buffer was added, and the BLA activity was measured asdescribed above.

The BLA activity that was determined by the CEA-binding assay and thetotal BLA activity found in the lysate plates were compared and variantswere identified which showed high levels of total BLA activity and highlevels of CEA-binding activities.

The winners were confirmed in 4 replicates using a similar protocol: thewinners were cultured in 2 ml of LB containing 5 mg/l chloramphenicoland 0.1 mg/l cefotaxime for 3 days. Protein was released from the cellsusing BPER reagent. The binding assay was performed as described abovebut different dilutions of culture lysate were tested for each variant.Thus one can generate a binding curve which provides a measure of thebinding affinity of the variant for the target CEA. FIG. 11A showsbinding curves. Culture supernatants were also analyzed by SDSpolyacrylamid electrophoresis. FIG. 11B shows the electropherogram of 7variants from NA05. The band of the fusion protein is labeled forvariant NA05.6. Table 6 shows a ranking of 6 variants. The data clearlyshow that NA05.6 produces significantly larger quantities of fusionprotein compared to the fusion construct pME27.1.

Table 6 showing the sequence of 6 variants with the largest improvementin stability: clone mutations NA05.6 R13K, T16G, W181V NA05.8 R13K,F170Y, A234G NA05.9 K3Q, S14P, L37V, E42G, E136Q, M146V, W181V, A234GNA05.10 K3Q, L37V, P170Y, W181V NA05.12 K3Q, S14P, L37V, M146V NA05.15M146V, F170Y, A194D

E. Construction of Library NA06

Clone NA05.6 was chosen as the best variant and was used as the templatefor a second round of combinatorial mutagenesis. We used a subset of thesame mutagenic primers that had been used to generate library NA05 togenerate combinatorial variants with the following mutations: K3Q, L37V,E42G, E136Q, M146V, F170Y, A194D, A234G which had been identified inother winners from library NA05. We did not use the primer encodingmutation S14P as its sequence overlapped with mutations R13K and T16Gthat are present in NA05.6. A combinatorial library was constructedusing QuikChange Multisite as described above and was called NA06.Template was pNA05.6 and 1 μl of primers mix (10 μM stock of all primerscombined containing 1.25 μM each primer) were used.

F. Screening of Library NA06

The screen was performed as described above with the followingmodifications:

291 variants were screened on 3 96-well plates. 10 μl sample from thelysate plates was added to 180 μl of 10 μg/ml thermolysin (Sigma) in 50mM imidazole buffer pH 7.0 containing 0.005% Tween-20 and 10 mM calciumchloride. This mixture was incubated for 1 h at 37 C to hydrolyzeunstable variants of NA05.6. This protease-treated sample was used toperform the CEA-binding assay as described above.

Promising variants were cultured in 2 ml medium as described above andbinding curves were obtained for samples after thermolysin treatments.FIG. 11C shows binding curves for selected clones. It can be seen that anumber of variants retain much more binding activity after thermolysinincubation than the parent NA05.6.

Table 7 showing 6 variants which are significantly more proteaseresistant than NA05.6: Clone mutations NA06.2 R13K, T16G, W181V, L37V,E42G, A194D NA06.4 R13K, T16G, W181V, L37V, M146V NA06.6 R13K, T16G,W181V, L37V, M146V, K3Q NA06.10 R13K, T16G, W181V, L37V, M146V, A194DNA06.11 R13K, T16G, W181V, L37V, K3Q, A194D NA06.12 R13K, T16G, W181V,L37V, E136QAll 6 variants have the mutation L37V which was rare in randomly chosenclones from the same library. Further testing showed that variant NA06.6had the highest level of total BLA activity and the highest proteaseresistance of all variants.

Example 4 Generation of an scFV that has pH-Dependent Binding

A. Choosing Positions for Mutagenesis

The 3D structure of the scFv portion of NA06.6 was modeled based on thepublished crystal structure of a close homologue, MFE-23 [Boehm, M. K.,A. L. Corper, T. Wan, M. K. Sohi, B. J. Sutton, J. D. Thornton, P. A.Keep, K. A. Chester, R. H. Begent and S. J. Perkins (2000) Biochem J 346Pt 2, 519-28, Crystal structure of the anti-(carcinoembryonic antigen)single-chain Fv antibody MFE-23 and a model for antigen binding based onintermolecular contacts] using the software package MOE (ChemicalComputing Group, Montreal, Canada) and using default parameters. A spacefilling model of the structure was visually inspected. Side chains inthe CDRs were ranked as follows: 0=burried; 1=partially exposed;2=completely exposed. Side chain distance to CDR3 was ranked as: 0=sidechain is in CDR3; 1=side chain is one amino acid away from CDR3; 2=sidechain is two amino acids away from CDR3. In a few cases, residuesflanking the CDRs were included if they fit the distance and exposurecriteria.

Based on this ranking, the following side chains were targeted formutagenesis:

-   a) exposure=2 and distance=2 or smaller-   b) exposure=1 and distance<2-   40 positions in the CDRs matched these criteria.-   FIG. 14 shows the CDRs and the residues that were chosen for    mutagenesis.

The table below shows the criteria and position of the 40 sites thatwere chosen for mutagenesis.

B. Construction of Library NA08

A combinatorial library was constructed where the 40 selected positionswere randomly replaced with aspartate or histidine. The substitutionswere chosen as it has been reported that ionic interactions betweenhistidine side chains and carboxyl groups form the structural basis forthe pH-dependence of the interaction between IgG molecules and the Fcreceptor [Vaughn, D. E. and P. J. Bjorkman (1998) Structure 6, 63-73,Structural basis of pH-dependent antibody binding by the neonatal Fcreceptor].

The QuikChange multi site-directed mutagenesis kit (QCMS; StratageneCatalog # 200514) was used to construct the combinatorial library NA08using 40 mutagenic primers. The primers were designed so that they had17 bases flanking each side of the codon of interest based on thetemplate plasmid NA06.6. The codon of interest was changed to thedegenerate codon SAT to encode for aspartate and histidine. All primerswere designed to anneal to the same strand of the template DNA (i.e.,all were forward primers in this case). The QCMS reaction was carriedout as described in the QCMS manual with the exception of the primerconcentration used; the manual recommends using 50-100 ng of each primerin the reaction, whereas significantly lower amounts of each primer wereused in this library as this results in a lower parent templatebackground. In particular, 0.4 μM of all primers together were used. Theindividual degenerate primer concentration in the final reaction was0.01 μM (approximately 2.5 ng).

The QCMS reaction contained 50-100 ng template plasmid (NA06.6, 5178bp), 1 μl of primers mix (10 μM stock of all primers to give the desiredprimer concentration mentioned above), 1 μl dNTPs (QCMS kit), 2.5 μl 10×QCMS reaction buffer, 18.5 μl deoinized water, and 1 μl enzyme blend(QCMS kit), for a total volume of 25 μl. The thermocycling program was 1cycle at 95° for 1 min., followed by 30 cycles of 95° C. for 1 min., 55°C. for 1 min., and 65° C. for 10 minutes. DpnI digestion was performedby adding 1 μl DpnI (provided in the QCMS kit), incubation at 37° C. for2 hours, addition of 0.5 μl DpnI, and incubation at 37° C. for anadditional 2 hours. 1 μl of each reaction was transformed into 50 μl ofTOP10 electrocompetent cells from Invitrogen. 250 μl of SOC was addedafter electroporation, followed by a 1 hr incubation with shaking at 37°C. Thereafter, 10-50 μl of the transformation mix was plated on LAplates with 5 ppm chloramphenicol (CMP) or LA plates with 5 ppm CMP and0.1 ppm of cefotaxime (CTX) for selection of active BLA clones. Thenumber of colonies obtained on both types of plates was comparable (652on the CMP plate and 596 colonies on the CMP+CTX plate for 10 μl of thetransformation mix plated). Active BLA clones from the CMP+CTX plateswere used for screening, whereas random library clones from the CMPplates were sequenced to assess the quality of the library.

Primers for the reaction are shown in Table 8. TABLE 8 Primers for CDRs:position distance to residue CDRs exposure CDR3 primer sequence K 30 2 2cttctggcttcaacattsatgactcctatatgcactg D H1 31 2 1ctggcttcaacattaaasattcctatatgcactgggt S H1 32 1 1gcttcaacattaaagacsattatatgcactgggtgag Y H1 33 2 1tcaacattaaagactccsatatgcactgggtgaggca H H1 35 1 1ttaaagactcctatatgsattgggtgaggcaggggcc W H2 50 2 1gcctggagtggattggasatattgatcctgagaatgg D H2 52 2 2agtggattggatggattsatcctgagaatggtgatac E H2 54 2 2ttggatggattgatcctsataatggtgatactgaata N H2 55 2 2gatggattgatcctgagsatggtgatactgaatatgc D H2 57 2 1ttgatcctgagaatggtsatactgaatatgccccgaa T H2 58 1 1atcctgagaatggtgatsatgaatatgccccgaagtt E H2 59 2 1ctgagaatggtgatactsattatgccccgaagttcca P H2 62 2 1gtgatactgaatatgccsataagttccagggcaaggc K H2 63 2 3atactgaatatgccccgsatttccagggcaaggccac Q H2 65 2 2aatatgccccgaagttcsatggcaaggccacttttac E 98 1 0ccgtctattattgtaatsatgggactccgactgggcc G 99 1 0tctattattgtaatgagsatactccgactgggccgta T H3 100 2 0attattgtaatgaggggsatccgactgggccgtacta P H3 101 2 0attgtaatgaggggactsatactgggccgtactactt T H3 102 2 0gtaatgaggggactccgsatgggccgtactactttga G H3 103 2 0atgaggggactccgactsatccgtactactttgacta P H3 104 2 0aggggactccgactgggsattactactttgactactg Y H3 106 2 0ctccgactgggccgtacsattttgactactggggcca S L1 162 2 2taacctgcagtgccagcsatagtgtaagttacatgca S L1 163 2 1cctgcagtgccagctcasatgtaagttacatgcactg V L1 164 1 1gcagtgccagctcaagtsatagttacatgcactggtt S L1 165 2 1gtgccagctcaagtgtasattacatgcactggttcca Y L1 166 2 1ccagctcaagtgtaagtsatatgcactggttccagca Y 183 1 0ctcccaaactcgtgattsatagcacatccaacctggc S L2 184 2 0ccaaactcgtgatttatsatacatccaacctggcttc T L2 185 1 1aactcgtgatttatagcsattccaacctggcttctgg S L2 186 2 2tcgtgatttatagcacasataacctggcttctggagt N L2 187 2 1tgatttatagcacatccsatctggcttctggagtccc A L2 189 1 1atagcacatccaacctgsattctggagtccctgctcg S L2 190 2 1gcacatccaacctggctsatggagtccctgctcgctt R L3 225 2 2cttattactgccagcaasattctagttacccactcac S L3 226 2 2attactgccagcaaagasatagttacccactcacgt S L3 227 1 2actgccagcaaagatctsattacccactcacgttcg Y L3 228 1 2gccagcaaagatctagtsatccactcacgttcggtg L L3 230 1 2aaagatctagttacccasatacgttcggtgctggcac

C. Sequencing of Variants

Variants were grown overnight with shaking at 37° C. in 5 mL cultures ofLA containing 5 ppm of CMP. Miniprep DNA was prepared using a Qiagen kitand the BLA gene within each clone was sequenced using the M13 reverseand nsa154f primers. M13 reverse: CAGGAAACAGCTATGAC nsa154f:GGACCACGGTCACCGTCTCCTC

D. Screen pH-Dependent Binding

Library NA08 was plated onto agar plates with LA medium containing 5mg/l chloramphenicol and 0.1 mg/l cefotaxime (Sigma). 552 colonies weretransferred into a total of six 96-well plates containing 100 ul/well ofLA medium containing 5 mg/l chloramphenicol and 0.1 mg/l cefotaxime.Four wells in each plate were inoculated with TOP10/NA06.6 as areference. The plates were grown overnight at 37 C. The next day thecultures were used to inoculate fresh plates (production plates)containing 100 ul of the same medium using a transfer stamping tool andglycerol was added to the master plates which were stored at −70 C. Theproduction plates were incubated in a humidified shaker at 37 C for 2days. 100 ul of BPER (Pierce, Rockford, Ill.) per well was added to theproduction plates to release protein from the cells. The productionplates were diluted 100-fold in PBST (PBS containing 0.125% Tween-20)and BLA activity was measured by transferring 20 ul diluted lysate into180 ul of nitrocefin assay buffer (0.1 mg/ml nitrocefin in 50 mM PBSbuffer containing 0.125% octylglucopyranoside (Sigma)) and the BLAactivity was determined at 490 nm using a Spectramax plus plate reader(Molecular Devices, Sunnyvale, Calif.).

Binding to CEA (carcinoembryonic antigen, Biodesign Intl., Saco, Me.)was measured using the following procedure: 96-well plates were coatedwith 100 ul per well of 5 ug/ml of CEA in 50 mM carbonate buffer pH 9.6overnight. The plates were washed with PBST and blocked for 1-2 hourswith 300 ul of casein (Pierce, Rockford, Ill.). 100 ul of sample fromthe production plate diluted 100-1000 fold was added to the CEA coatedplate and the plates were incubated for 2 h at room temperature.Subsequently, the plates were washed four times with PBST and 200 ulnitrocefin assay buffer was added, and the BLA activity was measured asdescribed above. CEA binding was measured in 50 mM phosphate buffer pH6.5 and in a separate experiment in 50 mM phosphate buffer pH 7.4.

The BLA activity that was determined by the CEA-binding assay at pHs of6.5 and 7.4, and the total BLA activity found in the lysate plates werecompared and variants were identified which showed good binding to CEAat pH 6.5 but significantly weaker binding at pH 6.5. A comparison ofthe binding at pH6.5 versus pH 7.4 is shown in FIG. 13.

Winners were confirmed by culturing them in 5 ml of LB medium containing5 mg/l chloramphenicol and 0.1 mg/l cefotaxime (Sigma) for 2 days at 37C. Subsequently, the cultures were centrifuged and the pellet wassuspended in 375 ul of BPER reagent to release the fusion protein. TheBLA activity in each sample was determined by transferring 20 ul of theappropriately diluted sample to 180 ul of 180 ul of nitrocefin assaybuffer (0.1 mg/ml nitrocefin in 50 mM PBS buffer containing 0.125%octylglucopyranoside (Sigma)) and the absorbance at 490 nm wasmonitored. One unit of activity was defined as the amount of BLA thatleads to an absorbance increase of one mOD per minute. The samples werediluted based on their total content of BLA activity and the CEA-bindingassay was performed as described above but adding various sampledilutions to each well.

Thus, one can obtain binding curves for each sample that reflect theaffinity of the variants to CEA. FIG. 15 shows CEA-binding curvesmeasured at pH 7.4 and pH 6.5 for several variants of interest. All 5variants show increased pH-dependence of CEA binding. Whereas, theparent NA06.6 binds only slightly better at pH 6.5 compared to pH 7.4,some of the variant show much stronger binding to CEA at pH 6.5 comparedto pH 7.4. Of particular interest are variants NA08.15 and NA08.17 whichshow very weak binding to CEA at pH 7.4 but significant binding at pH6.5.

Table 9, below, shows variants with the greatest binding improvement.TABLE 9 clone mutations NA08.1 W50H, Y166D NA08.3 S190D, S226D NA08.4S190D, T100D NA08.9 Y166D NA08.12 T102H, Y166D, S226D NA08.13 Q65H,S184D, S226D NA08.14 P101D NA08.15 S184D, S226D NA08.17 S184D, W50HNA08.24 T102D, S226D NA08.45 T102D, Y166D NA08.51 P104H, Y166D NA08.64Q65D, Y166D

Example 5 Purification of ME27.1

Purification of ME27.1 from cell extract was done using Cation ExchangeChromatography. This was performed with the aid of a high performanceliquid chromatographic system (AKTA™explorer 10, Amersham Biosciences)on a 7.3 mL CM Ceramic HyperF cation exchange column (CiphergenBiosystems). The column was first equilibrated withloading/equilibration buffer. The prepared ME27.1 extract was applied tothe column at 300 cm/hr, followed by washing with the equilibrationbuffer. The bound proteins were eluted using a sodium chloride gradient.The eluted fractions were analyzed using colorimetric activity assay(o-nitrocefan as substrate) and 4-12% Bis-Tris SDS-PAGE reducing gelwith MES running buffer (Novex).

Buffers

The following buffers were used for the purification using CationExchange Chromatography:

-   Loading/Equilibration: 50 mM Sodium Acetate, pH 5-   Elution: 50 mM Sodium Acetate containing 1M Sodium Chloride, pH 5-   Regeneration: 0.5M Sodium Hydroxide and 1M Sodium Acetate, pH 4

a) Extract Sample Preparation

E. coli cells containing gene of interest were cultured in TB broth. Theproduction media flasks were incubated at 30° C., 150-200 rpm for 18-24hours. After fermentation, the E. coli cells were centrifuged at 4,000rpm for 30 minutes. The supernatant was discarded and BPER detergent(Pierce product #78266) was added to the pellet to lyse the cells (27 mLof B-PER per gram of cell pellet). The lysed cells were centrifuged at18,000 rpm for 20 minutes to remove cell debris. The supernatantcontaining the ME27.1, referred to as the “extract”, was used for thesubsequent purification experiments. The extract was stored at 4° C.until use for subsequent purification experiments.

The following sample pretreatment steps were followed to prepare theextract for subsequent chromatography purification experiments.

-   Dilute ME27.1 extract (conductivity 9.5 mS/cm and pH 7.3) with 1    part of equilibration buffer (see below). pH of the diluted extract    was 6.51.-   Adjust pH to 5.0 using ˜20% acetic acid.-   Filter pH adjusted extract through 0.2 μm filter unit with ˜1%    diatomaceous earth as admix.

FIG. 16 shows the overall the chromatogram obtained using the 18 hoursold extract. The fractions from peak #1 constitute 23% of the totaleluted activity, with the major protein band near molecular weight (MW)equivalent to the ME27.1 MW (see FIG. 17, gel on left). Peaks #2, #3 and#4 constitute 76.6% of the total eluted activity but SDS-PAGE gel (seeFIG. 17, gel on right) shows that these fractions contains relativelysmall amount of protein near ME27.1 MW. There was an increasing amountprotein bands at MW below ME27.1 MW.

FIG. 18 shows the overall chromatogram obtained using 26 hours oldextract. The chromatogram is significantly different from FIG. 16. Therelative proportion of peak #1 to peak #2 has decreased significantlyfor the 26 hours extract compared to the 18 hour extract. SDS-PAGE gelsshows that the small peak #1 contains mostly protein near ME27.1 MW butthe remainder fractions (from peak #2 and onwards) contain mostlyprotein bands with MW lower that ME27.1 (see FIG. 19).

FIG. 20 shows the overall chromatogram obtained using >>26 hours old(4-5 days) extract. The chromatogram is significantly different fromFIG. 16 and FIG. 18. The distinct peak #1 found in 18 hrs (FIG. 16) and26 hours (FIG. 18) old extract has collapsed into a shoulder ofequivalent peak #2 found in FIG. 16 and FIG. 18. SDS-PAGE gels showsthat the shoulder contains two main bands, one near the ME27.1 MW andthe other at lower MW. The main peak #1 fractions contain 88% of theactivity eluted, and yet the main protein band is below the MW ofME27.1. Mass spec analysis confirmed that the lower band is degradedME27.1 (see FIG. 21, left gel circled band).

Purification of NA05.6 extract was done using Anion ExchangeChromatography follow by Affinity chromatography usingaminophenylboronic acid (PBA) resin.

The anion exchange chromatography was performed with the aid of a highperformance liquid chromatographic system (AKTA™explorer 10, AmershamBiosciences) on a 7 mL Poros HQ anion exchange column (PE Biosystems).The column was first equilibrated with loading/equilibration buffer. Theprepared NA05.6 extract was applied to the column at 300 cm/hr. TheNA05.6 was collected as the flow through and wash fractions. Thesefractions were analyzed for activity using colorimetric assay(o-nitrocefan as substrate) and purity using 4-12% Bis-Tris SDS-PAGEreducing gel with MES running buffer (Novex).

The PBA step was done using a 5 mL column containing PBA resin (SigmaA-8530 m-aminophenylboronic acid resin). The column was firstequilibrated with 20 mL of each equilibration buffers by gravity flow.The anion exchange partially purified flow through was used as the PBAfeed. The feed was applied to the column by gravity flow and wash withwash buffer. The bound sample was eluted with elution buffer. Sampleswere analyzed for activity using colorimetric assay (o-nitrocefan assubstrate) and purity using 4-12% Bis-Tris SDS-PAGE reducing gel withMES running buffer (Novex).

Buffers

The following buffers were used for the purification using AnionExchange Chromatography:

-   Load/Equilibration: 50 mM Tris, pH 7.4-   Regeneration: 0.5M NaOH

The following buffers were used for the purification using PBA AffinityChromatography:

-   Equilibration Buffers: 0.5 M sorbitol with 1 M NaCl; 0.5 M Borate at    pH 7; and 20 mM TEA with 0.5 M NaCl, pH 7-   Wash buffer: 20 mM TEA with 0.5M NaCl-   Elution Buffer: 0.5 M borate with 0.5M NaCl

b) Extract Sample Preparation for Anion Exchange Chromatography

The flow through contains 79% of the activity loaded onto the anionexchange column. Subsequent purification of the flow through fractionusing PBA affinity chromatography shows that all the protein eluted fromcontain protein near NA05.6 MW (See FIG. 22) and 80% of the loadedactivity was recovered. This indicates that all the NA05.6 molecule isintact.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method of improving a binding characteristic of a binding sequencefor a target comprising: a) contacting the target with a reporter fusionunder conditions that allow the reporter fusion to bind to the target,wherein the reporter fusion comprises a reporter sequence and a variantbinding sequence that is derived from a prototype binding sequence thatbinds the target, and b) selecting the reporter fusion if it has animproved binding characteristic compared to the prototype bindingsequence.
 2. The method of claim 1 wherein the selected reporter fusionbinds to the target with an affinity that is greater than the bindingaffinity of the prototype binding sequence for the target.
 3. The methodof claim 1 wherein the selected reporter fusion binds to the target witha greater specificity than the prototype binding sequence has for thetarget.
 4. The method of claim 1, wherein step (b) comprises incubatingthe reporter fusion in the presence of proteases and/or under conditionswhich degrade or destabilize the reporter fusion.
 5. The method of claim4, wherein the conditions are at least one of heat, pH or incubation inthe presence of solutes that affect stability.
 6. The method of claim 1further comprising repeating steps (a) and (b) one or more times,wherein the binding sequence of the reporter fusion selected in aprevious step (b) is the prototype binding sequence of the subsequentstep (a).
 7. The method according to claim 1 further comprising removingthe reporter sequence from the binding sequence of the reporter fusionselected in step (b).
 8. A method of improving a binding characteristicof a binding sequence for a target comprising: a) contacting a targetwith a library comprising a multiplicity of reporter fusions, underconditions that allow a reporter fusion to bind the target, wherein saidreporter fusions comprise a reporter sequence and a variant bindingsequence derived from a prototype binding sequence that binds thetarget, and b) selecting a reporter fusion bound to the target that hasan improved binding characteristic.
 9. The method of claim 8 wherein theselected reporter fusion binds to the target with an affinity that isgreater than the binding affinity of the prototype binding sequence forthe target.
 10. The method of claim 8 wherein the selected reporterfusion binds to the target with a greater specificity than the prototypebinding sequence has for the target.
 11. The method of claim 8 furthercomprising repeating steps (a) and (b) one or more times, wherein thebinding sequence of the reporter fusion selected in a previous step (b)is the prototype binding sequence of the subsequent step (a).
 12. Themethod according to claim 8 further comprising removing the reportersequence from the binding sequence of the reporter fusion selected instep (b).
 13. A method of improving the binding affinity of a bindingsequence for a target comprising: a) making a reporter fusion bycovalently linking a prototype binding sequence to a reporter sequence,b) modifying the binding sequence to produce a variant binding sequence,c) contacting the target with the reporter fusion under conditions thatallow the reporter fusion to bind the target, and d) selecting thereporter fusion if it has an improved binding characteristic.
 14. Themethod of claim 13 wherein the selected reporter fusion binds to thetarget with an affinity that is greater than the binding affinity of theprototype binding sequence for the target.
 15. The method of claim 13wherein the selected reporter fusion binds to the target with a greaterspecificity than the prototype binding sequence has for the target. 16.The method according to claim 13 further comprising repeating steps (a)through (d) one or more times, wherein the variant binding sequence of areporter fusion selected in a previous step (d) is the prototype bindingsequence of the subsequent step (a).
 17. The method according to claim13 further comprising removing the reporter sequence from the bindingsequence of the reporter fusion selected in step (d).